Expression of SNARE proteins in enteroendocrine cell lines and functional role of tetanus
KeyMedicalInnovations：医学创新的关键
Since its founding in 1919, Olympus, a precision technology leader, creating innovative opto-digital solutions in healthcare, life science and consumer electronics products, has led the industry in developing medical innovations across its business lines. Throughout its history, Olympus has been known for pioneering many of the world’s firsts, such as the first gastrocamera, the first DNA computer for gene analysis and the first endoscope system featuring high-definition and Narrow Band Imaging™ technologies.Today, Olympus has a leading market share of more than 70% in the global medical endoscope business.Timeline of Innovation1927 Olympus pioneers affordable, high-quality biological microscope in Japan.The Showa GK biological microscope met the need for an affordable, high-quality, practical oilimmersion type microscope (microscope requiring a drop of oil between specimen and objectivelens for observations). For the first time, scientists could visualize the microscopic world. TheShowa GK biological microscope was at the pinnacle of microscopes produced in Japan at thetime.Since the launch of its first model, Olympus microscopes have become vital to observation atthe submicron level and are found in the leading research laboratories, clinical centers andeducational institutions worldwide. Today, Olympus clinical laboratory microscopes are used inthe U.S. more than any other brand.1950 Olympus develops world’s first gastrocamera.Mounted at the tip of a flexible tube, Olympus’ miniature camera could record the stomach on film. Thisrevolutionized endoscopy. For the first time, physicians could diagnose stomach conditions withoutperforming surgery, thus increasing the speed of diagnosis. For patients, the gastrocamera made theprocedure less invasive and decreased the risk of infection.1964 Olympus GTF gastrocamera fiberscope advances minimally invasive endoscopy.The 1960s saw another Olympus breakthrough with a fiberscope that allowed direct, real-timeobservation of the stomach that could transmit images in real time as well. This laid the foundation forminimally invasive endoscopic treatment. Setting the stage for further innovations, Olympusminiaturization technologies (once used for observation) have become the centerpiece of minimallyinvasive surgery, making treatment less invasive, shorter, less costly and more effective.1969 Physician and Olympus engineer revolutionize endoscopy with invention of device for nonsurgical removal of colon polyps.The polypectomy snare, for the first time, enabled physicians to nonsurgically remove polyps detected inthe colon during a colonoscopy. The invention resulted from the visionary collaboration between aphysician and an Olympus engineer. Considered by some as risky at the time, the polypectomy snaretoday is widely used in colorectal cancer screening. Colorectal cancer is one of the most preventablecancers when polyps are detected and removed early.1978 Olympus launches world’s first fully automated system in Japan to test blood for a broad spectrum of diseases.Olympus’ Automated Electrophoresis System (AES) for the first time entirely automated a complexlaboratory blood test for a broad range of disease stages. The AES increased lab productivity forperforming the test to separate antibody proteins in the blood serum called globulins. Prior to the launchof the AES, the test involved multiple steps, and highly skilled operators were required to handle themany requisite chemicals and procedures.(Procedures include serum application, electrophoretic run,staining, destaining, drying, densitometry, and recording.)Olympus’ AES responded to laboratories’ needs for fully automated electrophoresis systems amidincreasing test volume and built on Olympus’ development of a fully automated clinical chemistryanalyzer in 1969.d evelops world's first DNA computer for gene analysis.2002 OlympusIn Japan, Olympus unveiled the world's first functional computer for gene analysis, combining hugecomputing power and parallel processing. The result is a high-speed, fully automated process –from sample injection to reaction – that enables quantitative gene expression profiling for researchand medical fields, such as genetic diagnosis and drug discovery. This computer is expected to bethe fastest gene analyzer in the world.In the future, Olympus' technology may enable customized drug development for targetpopulations, rather than today's mass-demand approach.2005 Olympus introduces revolutionary system to simplify and streamline complexendoscopic procedure.The V-System, which incorporates a duodendoscope and EndoTherapy devices from Olympus,was developed to help simplify and streamline a therapeutic procedure known as EndoscopicRetrograde Cholangio-Pancreatography (ERCP). Conducted to correct a problem in the biliaryand/or pancreatic ducts, ERCP has traditionally been a time consuming and complicatedprocedure, where cooperation and coordination between the nurse and doctor is critical. The V-System’s design gives the endoscopist greater device control and the flexibility to give control ofspecific devices to the assistant.2005 Olympus unveils world’s first endoscope platform featuring high-definition andNarrow Band Imaging ™ technologies.The Olympus EVIS EXERA II™ Series 180 high-definition platform is the world’s first to deliverboth high-definition (HDTV) and Narrow Band Imaging (NBI) technologies. The HDTV signal,when used together with Olympus’ 180 series high-definition, NBI endoscopes, offersgastroenterologists remarkably clear views of anatomical structures and fine capillaries.Olympus’ NBI technology enhances visualization of the capillary network and mucosalmorphology during endoscopic observations of the gastrointestinal tract.2005Olympus presents world’s first 360-degree electronic radial scanning gastrovideoscope, expanding the potential of endoscopic ultrasound.The Olympus GF-UE160-AL5 is the world’s first 360-degree endoscope featuring electronic radialscanning. Combining exceptional scope capability with advanced ultrasound image quality andfunctionality, it allows for enhanced diagnostic capability and easier orientation in the upper GI tract.Additionally, the scope’s wide angulation range results in outstanding maneuverability supportingexamination of the surface of the gastrointestinal tract as well as the layers beyond the wall, enablingphysicians to determine the extent to which a lesion has penetrated.2006 Olympus commercializes the world’s first fully autoclavable flexible bronchovideoscope.The introduction of the Olympus BF-Q180-AC provides hospitals and bronchoscopists with animaging quality new to bronchoscopy while delivering sterilization benefits of autoclaving. Proprietarymaterials representing a new standard in durability along with advanced engineering enable thisscope to withstand heat while maintaining its signature Olympus performance characteristics.2007 Olympus advances capsule endoscopy with the first commercially available EndoCapsule with real-time viewer.Olympus’ high-resolution capsule endoscopy system redefines visualization of the small bowelmucosa. Endo Capsule is part of Olympus’ “EnteroPro” brand, the first total solution of its kind offeredby one manufacturer designed to help physicians diagnose and treat small bowel abnormalities. TheEndo Capsule offers unique features that provide physicians with exceptional imaging capabilities ofthe small bowel, including enhanced depth of field, the widest field of view available for a capsule,advanced color reproduction, automatic brightness control and structure enhancement. It alsointroduces the first commercially available portable, lightweight real-time viewer which convenientlyallows physicians to verify its full functionality before the patient ingests the capsule.# # # # #。
2014. RSV感染时干扰素alphbeta受体信号扩增肺部促炎因子生成
Alpha/Beta Interferon Receptor Signaling Amplifies EarlyProinflammatory Cytokine Production in the Lung during Respiratory Syncytial Virus InfectionMichelle Goritzka,Lydia R.Durant,Catherine Pereira,Samira Salek-Ardakani,*Peter J.M.Openshaw,Cecilia JohanssonCentre for Respiratory Infections,Respiratory Infections Section,National Heart and Lung Institute,Faculty of Medicine,Imperial College London,London,United KingdomABSTRACTType I interferons(IFNs)are produced early upon virus infection and signal through the alpha/beta interferon(IFN-␣/)recep-tor(IFNAR)to induce genes that encode proteins important for limiting viral replication and directing immune responses.To investigate the extent to which type I IFNs play a role in the local regulation of inflammation in the airways,we examined their importance in early lung responses to infection with respiratory syncytial virus(RSV).IFNAR1-deficient(IFNAR1؊/؊)mice dis-played increased lung viral load and weight loss during RSV infection.As expected,expression of IFN-inducible genes was mark-edly reduced in the lungs of IFNAR1؊/؊mice.Surprisingly,we found that the levels of proinflammatory cytokines and chemo-kines in the lungs of RSV-infected mice were also greatly reduced in the absence of IFNAR signaling.Furthermore,low levels of proinflammatory cytokines were also detected in the lungs of IFNAR1؊/؊mice challenged with noninfectious innate immune stimuli such as selected Toll-like receptor(TLR)agonists.Finally,recombinant IFN-␣was sufficient to potentiate the produc-tion of inflammatory mediators in the lungs of wild-type mice challenged with innate immune stimuli.Thus,in addition to its well-known role in antiviral resistance,type I IFN receptor signaling acts as a central driver of early proinflammatory responses in the lung.Inhibiting the effects of type I IFNs may therefore be useful in dampening inflammation in lung diseases character-ized by enhanced inflammatory cytokine production.IMPORTANCEThe initial response to viral infection is characterized by the production of interferons(IFNs).One group of IFNs,the type I IFNs,are produced early upon virus infection and signal through the IFN-␣/receptor(IFNAR)to induce proteins important for limiting viral replication and directing immune responses.Here we examined the importance of type I IFNs in early re-sponses to respiratory syncytial virus(RSV).Our data suggest that type I IFN production and IFNAR receptor signaling not only induce an antiviral state but also serve to amplify proinflammatory responses in the respiratory tract.We also confirm this con-clusion in another model of acute inflammation induced by noninfectious stimuli.Ourfindings are of relevance to human dis-ease,as RSV is a major cause of infant bronchiolitis and polymorphisms in the IFN system are known to impact disease severity.I nfections at mucosal surfaces need to be managed carefully by the host in order to avoid damage to barrier functions.The pathogen needs to be eradicated rapidly,but inflammation must be tightly regulated to prevent detrimental effects on organ func-tion.Nowhere is this more evident than in the lung,where any excess cell infiltration or damage will markedly affect gas ex-change.The lung is a major site of infection by viruses,and the adverse effects of dysregulated lung inflammation have a very sig-nificant impact on human health.The initial response to viral infection in the lung and elsewhere is characterized by the production of interferons(IFNs).There are 3types of IFNs;type I IFNs(including alpha IFN[IFN-␣]and IFN-),type II IFNs(IFN-␥),and the recently discovered type III IFNs(IFN-).Irrespective of type,IFNs induce cell-intrinsic an-tiviral responses,activate natural killer(NK)cells,macrophages, and dendritic cells(DCs),and regulate innate and adaptive im-mune responses(1–3).IFN-␥is mainly produced by NK and T cells,while the synthesis of type I and type III IFNs,as well as other cytokines such as interleukin-6(IL-6)and tumor necrosis factor alpha(TNF-␣),is induced in immune and nonimmune cell types upon direct recognition of viral molecules by pattern recognition receptors(PRRs)such as Toll-like receptors(TLRs)and RIG-I-like receptors(RLRs)(1–4).The effect of IFN-is restricted to epithelial cells at mucosal surfaces,which express the relevant re-ceptor(1,2).In contrast,the receptor for type I IFNs,called IFNAR,is expressed ubiquitously by all cells.IFN-␣/is producedin the lung following infection with many viruses such as New-castle disease virus,influenza virus,respiratory syncytial virus(RSV),and human metapneumovirus(2,5–9).RSV is the major cause of infant bronchiolitis(10).While RSVdisease manifests as a simple common cold in the majority ofcases,between2and3%of children develop severe bronchiolitis.The variation in disease severity seems to be mostly due to hostReceived4February2014Accepted11March2014Published ahead of print19March2014Editor:T.S.DermodyAddress correspondence to Cecilia Johansson,c.johansson@.*Present address:Samira Salek-Ardakani,Leukocyte Biology Section,NationalHeart and Lung Institute,Imperial College London,London,United Kingdom.Copyright©2014Goritzka et al.This is an open-access article distributed underthe terms of the Creative Commons Attribution3.0Unported license.doi:10.1128/JVI.00333-14 Journal of Virology p.6128–6136June2014Volume88Number11rather than viral factors and has recently been associated with polymorphisms in several innate immunity genes,in particular many that control the IFN system(11–14).The IFN system may therefore be a key regulator of RSV-induced lung inflammation.To test the impact of type I IFNs on viral infections,IFNAR1-deficient(IFNAR1Ϫ/Ϫ)mice,which lack all signaling in response to IFN-␣/(15),have been widely used.For infections such as with reovirus or Chikungunya virus,loss of IFNAR signaling is detrimental,leading to overwhelming infection and death(16, 17).For some viruses(including influenza virus and RSV),the effect of IFNAR1deficiency is not as severe and does not impact on survival from infection(18,19).Whether type I IFNs are involved only in inducing an antiviral state in the lung or whether they have a more general effect in regulation of lung inflammation has not been fully elucidated. Here,we address this question by comparing inflammation in the lungs of wild-type and IFNAR1-deficient mice in response to chal-lenge with selected TLR agonists or RSV infection.Surprisingly,in all cases IFNAR deficiency was associated with a marked decrease in the production of proinflammatory cytokines and chemokines in the lung.Furthermore,type I IFN administration to the lung potentiated inflammation in mice.We suggest that type I IFN production and IFNAR receptor signaling not only induce an an-tiviral state but also serve to amplify proinflammatory responses in the respiratory tract.MATERIALS AND METHODSMice,virus stocks,TLR agonists,cytokines,and infection.Six-to10-week-old C57BL/6(Harlan or Charles River,United Kingdom)or IFNAR1Ϫ/Ϫmice on a C56BL/6background(obtained from C.Reis e Sousa,London Research Institute,London,United Kingdom)were main-tained under pathogen-free conditions under UK Home Office guide-lines.Plaque-purified human RSV(originally strain A2from the ATCC, United States)was grown in HEp-2cells(20).Age-and sex-matched mice were lightly anesthetized and infected intranasally(i.n.)with2ϫ106 focus-forming units(FFU)of RSV in100l.For lung challenge with innate stimuli,CpG(1.25g/g bodyweight),poly(I·C)HMW(high mo-lecular weight;3.5g/g),and lipopolysaccharide(LPS;500or50ng/g)in 100l were administered i.n.(all from Invivogen).Recombinant IFN-␣11(Miltenyi Biotech)was administered i.n.at500ng/mouse.Cell collection and preparation.Bronchoalveolar lavage(BAL)was carried out byflushing3times with1ml phosphate-buffered saline(PBS) containing0.5mM EDTA.For determination of cellular composition in the BALfluid,cells were transferred onto a microscope slide(Thermo Scientific)using a cytospin centrifuge and stained with hematoxylin and eosin(H&E;Reagena).Chemokine and cytokine detection.Chemokines and cytokines were quantified by a20-plex Luminex kit according to the manufacturer’s in-structions(Life Technologies),and data were acquired with a Bio-plex 200system(Bio-Rad laboratories,United Kingdom).The concentration of cytokines in each sample was determined according to the standard curve using the Bio-plex6software(Bio-Rad laboratories).IFN-2/3 (R&D),CXCL1(R&D),or IFN-␣(21)levels in the BALfluid were mea-sured by enzyme-linked immunosorbent assay(ELISA).Data were ac-quired on a SpectraMax Plus plate reader(Molecular Devices)and ana-lyzed using SoftMax software(version5.2).Gene expression.Lungs were homogenized in TRIzol(Invitrogen)using a TissueLyser LT(Miltenyi Biotech).RNA extractions were performed with chloroform-isopropanol.Removal of DNA from TRIzol-extracted RNA was carried out using the DNA-free kit according to the manufacturer’s manual (Ambion).RNA yields were determined by using a NanoDrop1000spectro-photometer.cDNA conversion was performed using a high-capacity RNA-to-cDNA kit according to the manufacturer’s instructions(Applied Biosystems).To quantify mRNA levels in lung tissue,real-time quantitative PCR(RT-qPCR)was performed.For mRNA analysis,the following primers and probes were used:for Tnfa,forward primer,5=-CATCTTCTCAAAATT CGAGTGACAA-3=,reverse primer,5=-TGGGAGTAGACAAGGTACAACC C-3=,and probe,5=-FAM-CACGTCGTAGCAAAC-TAMRA-3=;and for Ifng,forward primer,5=-TCAAGTGGCATAGATGTGGAAGAA-3=,reverse primer,5=-TGGCTCTGCAGGATTTTCATG-3=,and probe,5=-FAM-TCA CCATCCTTTTGCCAGTT-TAMRA-3=.Quantitative PCRs(qPCRs)for Ifnl2/3,Ifnb,Rsad2(Viperin),Oas1a,Eif2ak2(PKR),and L gene were per-formed using primers and probes previously described(20,22–24).The assay was performed using the Quantitect Probe PCR master mix(Qiagen)and the 7500Fast real-time PCR system(Applied Biosystems).For absolute quanti-fication,the exact number of copies of the gene of interest was calculated using a plasmid DNA standard curve for each gene.Results were normalized using Gapdh(encoding glyceraldehyde-3-phosphate dehydrogenase)as a housekeeping gene(Applied Biosystems).For relative quantification,the ex-pressions of Ddx58(RIG-I),Cxcl10,Cxcl1,Il6,Il1b,and Mx1(all from Ap-plied Biosystems)relative to the housekeeping gene Gapdh were determined. The⌬C T(cycle threshold difference)between the target gene and Gapdh was calculated for each sample and expressed as2Ϫ⌬Ct.Analyses were performed using7500Fast System SDS software(Applied Biosystems).Statistical analysis.Results are presented as meansϮstandard errors of the means(SEM).The significance of results between the groups was analyzed by two-tailed,nonparametric,unpaired Mann-Whitney t test (Prism software;Graph-Pad Software Inc.)and is indicated in thefigures as follows:*,PϽ0.05;**,PϽ0.01;***,PϽ0.001).P values ofϽ0.05were considered significant.RESULTSType I,II,and III interferon production is abrogated in IFNAR1؊/؊mice infected with RSV.In order to investigate the role of IFNAR signaling in lung inflammation induced by infec-tious challenge,wild-type(wt;C57BL/6)and IFNAR1-deficient (IFNAR1Ϫ/Ϫ)mice were infected i.n.with RSV.Wefirst assessed the effect of IFNAR deficiency on viral control.IFNAR1Ϫ/Ϫmice showed a significantly higher viral load in the lung,as measured by the copy number of viral L gene RNA,compared to wt mice from 8h postinfection(p.i.)until day14p.i.(the latest time point stud-ied;Fig.1A).A delay in viral clearance was also apparent,as11of 12IFNAR1Ϫ/Ϫmice had detectable L gene in the lungs at day14 pared to only1of12mice in the wt group(Fig.1A).The increase in lung viral load was accompanied by greater weight loss, a measure of infection severity(25):IFNAR1Ϫ/Ϫmice started to lose weight at day5,lost significantly more weight than infected wt mice,and had a slower recovery(Fig.1B).We then assessed the levels of IFNs in the lung early after in-fection by mRNA analysis and protein detection.IFN-␣in the BALfluid of wt mice was detected from8h p.i.,with peak pro-duction at12to18h p.i.In contrast,no IFN-␣was detectable in IFNAR1Ϫ/Ϫmice after RSV infection or in wt mice inoculated with UV-inactivated RSV(Fig.2A and data not shown).Both wt and IFNAR1Ϫ/Ϫmice displayed similar levels of IFN-mRNA at 4h p.i.,but this increased100-fold at later time points in wt but not IFNAR1-deficient mice(Fig.2B).This is consistent with the known IFNAR-dependent positive-feedback loop for type I IFN production(26).IFN-␥(mRNA or protein)was not detected in IFNAR1Ϫ/Ϫmice at any time point(Fig.2C),in contrast to wt mice,which showed IFN-␥levels peaking at12and18h p.i.for mRNA and protein,respectively.Furthermore,although expres-sion of IFN-mRNA was induced in IFNAR1Ϫ/Ϫmice,it was manifestly lower than in wt controls and did not result in detect-Proinflammatory Cytokines Depend on IFNAR1Signaling6129able protein in BAL fluid (Fig.2D ).These data confirm and extend a previous study showing that expression of type I IFNs is reduced in RSV-infected IFNAR1Ϫ/Ϫmice (9).We assessed whether the reduced levels of all IFNs inIFNAR1Ϫ/Ϫmice impacted the expression of selected interferon-stimulated genes (ISGs).CXCL10could not be detected at either the mRNA or the protein level in lungs of IFNAR1Ϫ/Ϫmice but was induced in wt mice (Fig.2E ).Ddx58(RIG-I)mRNA levelsA)Weight curveB)Viral loadDays Post Infection% o f o r i g i n a l w e i g h t10101010101010Days Post Infection L g e n e c o p i e s / μg R N AIFNAR1-/- RSVwt RSVIFNAR1-/-naive wt naiveFIG 1Viral load and weight loss after RSV infection.C57BL/6wt and IFNAR1Ϫ/Ϫmice were infected intranasally with 2ϫ106FFU of RSV.RNA was isolated from lung tissue,and after conversion into cDNA,copy numbers of RSV L gene RNA were determined using quantitative PCR (qPCR).(A)Levels of RSV L gene RNA in the lung tissue of wt and IFNAR1Ϫ/Ϫmice at different time points after RSV infection.Copy numbers were determined using a plasmid standard.Data shown are pooled data from 3individual experiments with 3to 5mice per group in each experiment.The dotted line represents the detection limit.ND,not detectable.(B)Weights of infected and noninfected mice were monitored daily and plotted as a percentage of weight on the day of infection (day 0).The data shown are representative of at least 3experiments with 3to 5miceper group in each experiment.Error bars indicate the SEM.***,P Յ0.001;**,P Յ0.01;*,P Յ0.05comparing RSV-infected wt with RSV-infected IFNAR1Ϫ/Ϫmice.##,P Յ0.01comparing RSV-infected wt with naive wt mice.F)RIG-I mRNAMx-1 mRNAG)E)CXCL10 mRNACXCL10 proteinD)IFN-λ mRNAIFN-λ proteinC)wtIFNAR1 -/-A)IFN-α proteinIFN-γ mRNAIFN-γ proteinTime Post Infection (hrs)I F N -α (U /m l )Time Post Infection (hrs)M x 1 r e l. e x p . t o G a p d hTime Post Infection (hrs)C x c l 10 r e l. ex p . t o G a p d hTime Post Infection (hrs)C X C L 10 (p g /m l )Time Post Infection (hrs)D d x 58 r e l. e x p . t o G a p d h10101010I f n g c o p i e s / μg R N ATime Post Infection (hrs)FIG 2Interferon responses after RSV infection of IFNAR1Ϫ/Ϫand wt mice.C57BL/6wt and IFNAR1Ϫ/Ϫmice were intranasally infected with 2ϫ106FFU of RSV.(A)The level of IFN-␣was determined in bronchoalveolar lavage (BAL)fluid by ELISA.(B)RNA was isolated from lungs,and gene expression levels of IFN-were determined by qPCR.(C to E)IFN-␥(C),IFN-(D),and interferon-stimulated gene (ISG)CXCL10(E)gene expression levels in lung tissue and protein production in BAL fluid were quantified.(F and G)RIG-I (Ddx58)(F)and Mx-1(G)mRNA was determined by qPCR.Gene expression relative to GAPDH was calculated for CXCL10,RIG-I,and Mx-1.For IFN-,IFN-,and IFN-␥,copy numbers were determined using a plasmid standard.The detection limit for all assays was 200copies and is represented by the dotted line.Data shown are representative of at least 2experiments with 4or 5mice per group.Error bars indicate the SEM.Significance when comparing RSV-infected wt with RSV-infected IFNAR1Ϫ/Ϫmice:**,P Յ0.01;*,P Յ0.05.Goritzka et al. Journal of Virologyincreased above basal expression at 4h p.i.in both IFNAR1Ϫ/Ϫand wt mice but continued to rise only in the wt mice until 12h p.i.(Fig.2F ).Similar results were obtained with Mx-1(Mx-1;Fig.2G ),Rsad2(Viperin),Oas1a (OAS1),and Eif2ak2(PKR)gene expression (data not shown).In sum,our data suggest that the loss of type I IFN receptor signaling results in decreased expression of all IFN types early after RSV infection and prevents appropriate induction of ISGs.This is associated with a failure to control RSV replication and clear the virus rapidly,leading to increased pathol-ogy (weight loss).Proinflammatory cytokine responses are diminished in IFNAR1؊/؊mice infected with RSV.To further evaluate the in-flammatory response early after RSV infection,we measured ad-ditional cytokines,including IL-6,IL-1,and TNF-␣.Very little,if any,mRNA encoding these cytokines could be detected in the lungs of RSV-infected IFNAR1Ϫ/Ϫmice (Fig.3A ).In contrast,such mRNAs were easily detectable in wt mice as early as 4h p.i.,with a peak of expression at 8h p.i.(Fig.3A ).Early infiltration of neutrophils did not differ between wt and IFNAR1Ϫ/Ϫmice (Fig.3B ),so we also analyzed the expression of CXCL1(KC),a known neutrophil attractant.CXCL1mRNA and protein were detected at similar levels in both wt and IFNAR1Ϫ/Ϫmice at early time points (Fig.3C ).However,later during the infection CXCL1levels were significantly higher in wt mice (Fig.3C ).Levels of proinflammatory cytokines and chemokines were additionally measured in BAL fluid using a multiplex ap-proach.IL-2,IL-4,IL-10,IL-13,and IL-17A were not detected in the airways of either wt or IFNAR1Ϫ/Ϫmice at any time point after infection (data not shown).For some cytokines(IL-6,IL-1,TNF-␣,and IL-12p40),there were measurable levels in IFNAR1Ϫ/Ϫmice at 4to 8h p.i.,but this was not comparable to the levels in wt mice,which were 5to 10times greater (Fig.4).For other cytokines and chemokines such as IL-1␣,granulocyte-macrophage colony-stimulating factor (GM-CSF),IL-5,IL-12p40,CXCL9,and CCL3,there was no or very little induction in the airways of IFNAR1Ϫ/Ϫmice at any time point,while wt mice showed substantial levels peaking at 8to 12h p.i.(Fig.4B ).Overall,these results indicate that the lack of type I IFN receptor signaling results in a marked reduc-tion in induction of proinflammatory mediators in lung and airways upon pulmonary viral infection.IFNAR1-deficient mice display reduced proinflammatory cytokine responses to lung challenge with TLR agonists.To in-vestigate whether similar results applied to noninfectious stim-ulation of the airways,we administered different innate im-mune stimuli intranasally (i.n.)to wt and IFNAR1Ϫ/Ϫmice.Mice were sacrificed 24h following administration of CpG,poly(I·C),or LPS,and lung cytokine expression was measured using qPCR and ELISA.We observed significantly reduced mRNA levels of IFN-in response to the TLR9agonist CpG and the TLR4agonist LPS and of CXCL10in response to all TLR agonists tested in IFNAR1Ϫ/Ϫmice (Fig.5A ).The expres-sion of proinflammatory cytokines was also quantified.De-creased induction of IL-6,IL-1,and TNF-␣mRNA was ob-served with the TLR3,RIG-I,and MDA-5agonist,poly(I·C),in IFNAR1Ϫ/Ϫmice.Moreover,a significant reduction in IL-6mRNA was seen in IFNAR1Ϫ/Ϫmice treated with CpG com-pared to wt mice (Fig.5A ).Similarly,LPS-dependent induc-A)B)IL-6IL-1βTNF-αCXCL1 mRNANeutrophilsC)CXCL1 proteinwtIFNAR1 -/-Time Post Infection (hrs)I l 6 r e l. e x p . t o G a p d hTime Post Infection (hrs)I l 1b r e l. e x p . t o G a p d h10101010Time Post Infection (hrs)T n f a c o p i e s / μg R N ADays Post Infection%o f B A L c e l l sTime Post Infection (hrs)C x c l 1 r e l. e xp . t o G a p d hTime Post Infection (hrs)C X C L 1 (n g /m l )FIG 3Diminished induction of proinflammatory cytokine mRNA in IFNAR1Ϫ/Ϫmice during RSV infection.C57BL/6wt and IFNAR1Ϫ/Ϫmice were intra-nasally infected with 2ϫ106FFU of RSV.(A)RNA was isolated from lungs,and gene expression levels of IL-6,IL-1-,and TNF-␣were determined by qPCR.(B)Percentage of neutrophils in the BAL fluid at indicated days postinfection.Data shown are pooled data from 3individual experiments with 3to 5mice per group in each experiment.(C)CXCL1(KC)gene expression levels in lung tissue and protein production in BAL fluid were quantified.Gene expression relative to GAPDH was calculated for IL-6,IL-1,and CXCL1.For TNF-␣,copy numbers were determined using a plasmid standard.Dotted lines represent the detection limit.Data shown are representative of at least 2experiments with 4or 5mice per group,or for CXCL1data shown are pooled data from 2individual experiments with 3to 5mice per group in each experiment.Error bars indicate the SEM.Significance when comparing RSV-infected wt with RSV-infected IFNAR1Ϫ/Ϫmice:***,P Յ0.001;**,P Յ0.01;*,P Յ0.05.Proinflammatory Cytokines Depend on IFNAR1SignalingJune 2014Volume 88Number 11 6131tion of IL-1mRNA was reduced in lungs of IFNAR1Ϫ/Ϫmice (Fig.5A ).A similar pattern was detected at the protein level:IFN-␣was not induced after poly(I·C)treatment,and IL-6was not induced by any of the TLR agonists in IFNAR1Ϫ/Ϫmice (Fig.5B ).Thus,the lack of type I IFN receptor decreases the lung inflammatory response provoked by innate stimulation of the airways.IFN-␣potentiates proinflammatory cytokine induction.The above results suggested that type I IFNs might potentiate proin-flammatory responses in the lung.To explicitly test this hypothe-sis,one representative type I IFN,IFN-␣11,was administered in-tranasally to mice concomitantly with innate immune challenge.The dose of IFN-␣was similar to a dose previously used to inhibit RSV infection (8),and LPS was chosen as the stimulus because itdoes not induce high expression of type I IFNs in the lung (data not shown).Mice that received both IFN-␣and a suboptimal dose of LPS showed increased lung expression of mRNAs encoding IL-6and TNF-␣(Fig.6A )or IFN-␥(data not shown)24h post-challenge compared to mice that received only LPS or IFN-␣.Interestingly,intranasal administration of IFN-␣alone was sufficient to induce an increase in IL-6,TNF-␣,IFN-␥,and IL-1mRNA at 12h (Fig.6B )and IL-6and TNF-␣mRNA in the lung at 24h (Fig.6A ).This was not observed in IFNAR1Ϫ/Ϫmice,indicating that the effect is dependent on signaling via IFNAR (Fig.6)and not due,for example,to a contaminant.We conclude that type I IFNs markedly potentiate acute proin-flammatory responses induced by innate immune stimulation of the airways.DISCUSSIONType I IFNs are produced early after viral infection as a first line of host defense.They act on all cell types via the ubiquitously ex-pressed IFNAR to induce increased expression of more than 300different genes whose products eventually interfere with viral rep-lication and viral spread,as well as lead to the initiation of immune responses (3,10).Previous studies addressing the role of type I IFNs in the lung have focused mainly on adaptive immunity (8,9,19,27,28).Thus,the importance of type I IFNs signaling for the early innate immune response in the lung remains elusive.In this study,we uncover a general role for IFNAR signaling in amplify-ing acute lung inflammatory responses to innate stimuli.Further,we provide in vivo evidence for an important role of IFNAR in innate resistance to RSV lung infection and show that signaling through IFNAR is necessary for coordinating the inflammatory response to the virus.Our data suggest that type I IFNs are pivotal contributors to lung inflammation.We anticipated that other IFNs might compensate for the lack of type I IFN signaling during RSV infection,as previously shown for influenza virus (9,29–31),but were surprised to find that our data did not support this hypothesis;neither IFN-nor IFN-␥was upregulated in the lungs of IFNAR1Ϫ/Ϫmice during the early stages of RSV infection.Instead,expression of all IFNs and ISGs was decreased in RSV-infected IFNAR1Ϫ/Ϫmice compared to wt controls.This suggests that type I IFNs are involved in controlling the expression of IFN-␣/,IFN-␥,IFN-,and ISGs during RSV infection.For IFN-␣/,this is expected because type I IFN pro-A)GM-CSFIL-5IL-1-αTime Post Infection (hrs)I L -12p 40 (p g /m l )Time Post Infection (hrs)C X C L 9 (p g /m l )Time Post Infection (hrs)C C L 3 (p g /m l )Time Post Infection (hrs)I L -1α (p g /m l )Time Post Infection (hrs)G M -C S F (p g /m l )Time Post Infection (hrs)I L -5 (p g /m l )Time Post Infection (hrs)I L -6 (p g /m l )B)IL-6TNF-αTime Post Infection (hrs)I L -1β (p g /m l )Time Post Infection (hrs)T N F -α (p g /m l )IL-1βFIG 4Reduced levels of proinflammatory cytokines in IFNAR1Ϫ/Ϫmice during RSV infection.C57BL/6wt and IFNAR1Ϫ/Ϫmice were intranasally infected with2ϫ106FFU of RSV.At different time points postinfection protein levels of IL-6,IL-1,TNF-␣,IL-1␣,GM-CSF,IL-5,IL-12p40,CXCL9,and CCL3production were determined in BAL fluid by Luminex.Data shown are representative of at least 2experiments with 4or 5mice per group.Error bars indicate the SEM.Significance when comparing RSV-infected wt with RSV-infected IFNAR1Ϫ/Ϫmice:**,P Յ0.01;*,P Յ0.05.Goritzka et al.6132Journal of Virologyduction relies on a positive-feedback loop through the type I IFN receptor (26).In addition,previous studies have shown that type I IFNs play a critical role in induction of IFN-␥gene expression through the activation of STAT4(32,33)or increased signalingthrough other cytokine receptors such as IFN-␥receptor by in-creased levels of STAT1(19,34).Furthermore,since the IFN re-sponses were reduced in IFNAR1Ϫ/Ϫmice,this resulted in a di-minished induction of ISGs as has previously been shown for TLR stimulation (35,36)and for bone marrow-derived DCs (BMDCs)stimulated with RSV (37).Therefore,type I IFN production with subsequent IFNAR signaling is a key component of the entire IFN response early after RSV infection.Surprisingly,our data indicate that type I IFN production and subsequent IFNAR signaling are also a key component of the en-tire inflammatory response.Indeed,we found that the induction of proinflammatory cytokines (e.g.,IL-6,IL-1,and TNF-␣)was abrogated in the lungs of IFNAR1Ϫ/Ϫmice after RSV infection.A similar pattern was seen in BAL fluid for a broader array of pro-inflammatory cytokines and chemokines.Furthermore,IFNAR deficiency decreased the induction of proinflammatory cytokines in response to airway challenge with different innate immune stimuli,and IFN-␣augmented the proinflammatory response to LPS stimulation.Also,IFN-␣alone given intranasally drove a rapid and transient induction of proinflammatory cytokines inA)B)N aCp G P o l y I :CL P SN aCp G P o l y I :CL P S1010101010I f n l c o p i e s /μg R N AIFNAR1-/-wtN aCp G P o l y I :CL P SN aCp G P o l y I :CL P SC x c l 10 r e l. e x p . t o G a p d hIFNAR1-/-wtN aCp GP o l y I :CL P SN aCp GP o l y I :CL P SI l 6 r e l. e x p . t o G a p d hIFNAR1-/-wtN aCp GP o l y I :CL P SN aCp GP o l y I :CL P SI l 1b r e l. e x p . t o G a p d hIFNAR1-/-wtN aCp G P o l y I :CL P SN aC p G P o l y I :CL P S101010T n f a c o p i e s / μg R N AIFNAR1-/-wtN aCp GP o l y I :CL P SN aCp GP o l y I :CL P STLR Agonist I F N -α (U /m l )IFNAR1-/-wtN aCP GP o l y I :CL P SN aCP GP o l y I :CL P SI L -6 (n g /m l )IFNAR1-/-wtIL-6 mRNAIL-1β mRNATNF-α mRNAIFN-λ mRNACXCL10 mRNAIL-6 proteinIFN-α proteinFIG 5Induction of cytokines after intranasal challenge of IFNAR1Ϫ/Ϫand wt mice with innate stimuli.C57BL/6wt and IFNAR1Ϫ/Ϫmice were intranasally challenged with TLR agonists [CpG,1.25g/g body weight;poly(I·C),3.5g/g body weight;LPS,500ng/g body weight].Lungs and BAL fluid were collected 24h postchallenge.(A)RNA was isolated from lungs,and gene expression levels of IFN-,CXCL10,IL-6,IL-1,and TNF-␣were determined by qPCR.Gene expression relative to GAPDH was calculated for IL-1,IL-6,and CXCL10.For IFN-and TNF-␣,copy numbers were determined using a plasmid standard.(B)IFN-␣and IL-6detected in the BAL fluid using ELISA.Data shown are pooled data from 2individual experiments with 4or 5mice per group in each experiment.Error bars indicate the SEM.Significance when comparing RSV-infected wt with RSV-infected IFNAR1Ϫ/Ϫmice:***,P Յ0.001;**,P Յ0.01;*,P Յ0.05.Proinflammatory Cytokines Depend on IFNAR1SignalingJune 2014Volume 88Number 11 6133。
中医药治疗胰腺纤维化的临床对策及研究进展
·综述·DOI： 10.3969/j.issn.1001-5256.2023.09.034中医药治疗胰腺纤维化的临床对策及研究进展纪晓丹1，龚彪1，李兴佳1，吕婵1，徐莹21 上海中医药大学附属曙光医院消化科，上海 201203；2 上海中医药大学教学实验中心，上海 201203通信作者：徐莹，******************（ORCID： 0000－0002－4645－3094）摘要：胰腺纤维化是慢性胰腺炎疾病发展不可逆的主要病理变化，目前临床针对胰腺纤维化的治疗仍缺乏疗效确切的药物。
本文总结了近年关于中医药治疗胰腺纤维化的临床策略及研究进展。
中医辨证胰腺纤维化涉及到的脏腑有肝、胆、脾、胃；病理因素与火、瘀血、痰湿相关；中药提取物抗胰腺纤维化的相关研究涉及的药物类别包括健脾类、化湿类及化瘀类等，中药方剂治疗胰腺纤维化的相关机制信号通路主要是干预胰腺星状细胞的激活。
以上研究为中医药对胰腺纤维化的预防、干预及防治并发症的深入探索提供了参考。
关键词：胰腺炎，慢性；纤维化；中医药疗法基金项目：国家自然基金青年科学基金项目（82004162）；上海市青年科技英才扬帆计划（20yf1449500）；上海中医药大学附属曙光医院“四明青年基金”（SGKJ－201924）Application of traditional Chinese medicine in treatment of pancreatic fibrosis：Clinical strategies and research advancesJI Xiaodan1，GONG Biao1，LI Xingjia1，LYU Chan1，XU Ying2.（1. Department of Gastroenterology，Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine，Shanghai 201203，China；2. Teaching and Experiment Center of Shanghai University of Traditional Chinese Medicine， Shanghai 201203， China）Corresponding author： XU Ying，******************（ORCID： 0000－0002－4645－3094）Abstract：Pancreatic fibrosis is the main irreversible pathological change during the progression of chronic pancreatitis， and at present，there is still a lack of effective drugs for the treatment of pancreatic fibrosis in clinical practice. This article summarizes the application of traditional Chinese medicine （TCM） in the treatment of pancreatic fibrosis in recent years from the aspects of clinical strategies and research advances. The TCM syndrome differentiation of pancreatic fibrosis involves the liver，gallbladder，spleen，and stomach，and pathological factors are associated with fire，blood stasis，and phlegm dampness. The research on the anti－pancreatic fibrosis effect of TCM extracts mainly involves spleen－strengthening，dampness－resolving， and blood stasis－resolving drugs， and intervention against the activation of pancreatic stellate cells is the main signaling pathway involved in the mechanism of TCM prescriptions in the treatment of pancreatic fibrosis. The above studies provide a reference for in－depth research on the application of TCM in the prevention and intervention of pancreatic fibrosis and the prevention and treatment of related complications.Key words：Pancreatitis， Chronic； Fibrosis； Traditional Chinese Medicine TherapyResearch funding：National Natural Science Fund for Youth （82004162）； Shanghai Young Science and Technology Talents Sailing Program （20yf1449500）；“Siming Youth Fund” of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine （SGKJ－201924）胰腺纤维化是慢性胰腺炎疾病发展的主要病理变化［1］，在临床上针对慢性胰腺炎的治疗主要以改善疼痛、预防其急性发作，纠正胰腺内外分泌功能不全及防治并发症为主［2］。
The Expression of MicroRNA and microRNA Clusters in the Aging Heart
The Expression of microRNA and microRNA Clusters in the Aging HeartXiaomin Zhang,Gohar Azhar,Jeanne Y.Wei *Donald W.Reynolds Department of Geriatrics,The University of Arkansas for Medical Sciences and Geriatric Research,Education and Clinical Center,Central Arkansas Veterans Healthcare System,Little Rock,Arkansas,United States of AmericaIntroductionAdult aging is a complex biological process associated with a progressive decline in the physiological and biochemical perfor-mance of individual tissues and organs,leading to increased susceptibility to age-related disease and functional senescence [1–4].The functional changes observed in the older heart are associated with morphological changes that occur during normal aging process,which is characterized by a loss of with subsequent hypertrophy of the remaining viable myocytes,and proliferation of cardiac fibroblasts.As myocytes are lost and fibroblasts continue to proliferate and produce collagen,the physical properties of the aging heart become altered [1,2].The functional and morphological changes in the older heart are accompanied by changes in cardiac gene expression,which include a -MHC,b -MHC,ANF,SERCA2etc.[5–10].Recent studies suggest that microRNAs (miRNAs)may play a role in the regulation of gene expression in the aging process [11–13].MiRNAs are short (20to 23-nucleotide),endogenous,single-stranded RNA molecules that regulate gene expression byhybridization to messenger RNAs (mRNAs)with the consequence of messenger RNA degradation or translational inhibition of targeted transcripts.Genes that encode for miRNA are distributed across chromosomes either individually,or in clusters,in which two or more miRNA genes are located within a short distance on the same segment of a chromosome.The miRNA gene is usually transcribed by RNA polymerase II into a primary miRNA (pri-miRNA)transcript,which is cleaved at a hairpin-stem by the nuclear microprocessor complex containing Drosha and DGCR8proteins,and generates hairpin-shaped pre-miRNA.The pre-miRNA is then exported from the nucleus to the cytoplasm,where it is cleaved by Dicer to form a 22nt double-stranded miRNA duplexes [14].Following their production,miRNA duplexes are sorted to confer association with specific members of the Argonaute (Ago)family of proteins,which function as the core of the RNA-induced silencing complex (RISC)[15–20].Post-transcriptional regulation of gene expression is mediated by the RISC complex,which uses one strand of the miRNA molecule (miR,the guide strand)to target relevant messenger RNAs.Once the messenger RNA is targeted bymiRNAs,the RISC is thought to inhibit protein production either through blocking translation or by reducing messenger RNAstability [21,22].Previously,the microRNA passenger strand (miR*or miRNA*)was thought to be degraded without significant biological impact on gene regulation.Recent studies,including deep-sequencing efforts,however,indicate that a large number of miR*s are not simply degraded,but rather,associate with Ago1and/or Ago2and are also capable of silencing target transcripts [15,16,18,19].To determine the impact of advancing age on microRNA expression in the heart,we examined the microRNA expression level in old versus young adult (YA)hearts.We found that the expression of 65microRNAs,including 11miRNAs clusters was significantly altered during adult aging.In addition,we found that major changes in the expression of miRNA occurred from the post-maturational period through adult aging.Our data suggest that transcriptional regulation of microRNA transcripts and altered expression of Argonaut proteins contribute to age-related changes in the expression of both miR and miR*strands.Materials and Methods Animal tissuesHealthy C57BL/6mice were obtained from colonies main-tained by the National Institute of Aging (NIA),the National Institutes of Health,under contractual agreement with Harlan Sprague-Dawley,Inc.(Harlan,IN).After euthanasia,the hearts were removed from mice and subjected to standard RNA isolation and histological procedures.Some heart tissue samples (4months and 24months)were obtained from the Aged Rodent Tissue Bank at NIA.For each time point,there were three independent biological replicates.The studies were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC)at Central Arkansas Veterans Healthcare System (IACUC #4-02-03)and in accordance with the NIH Guiding Principles for Research Involving Animals and Human Beings.Total RNA isolationAll RNA samples were first isolated from the mouse cardiac ventricles using UltraSpec RNA Isolation Reagent as previously described.To minimize mouse DNA contamination and enrich the small RNA fraction,the total RNA samples were purified using miRNeasy Mini kit (Qiagen)and RNase-free DNase I according to the manufacture’s instruction manual [23,24].MicroRNA arraysThe ventricular tissue samples used for the microRNA array analysis were obtained from healthy young adult (4-month-old)and old (24-month-old)C57BL6mice.The RNA sample isolation and microRNA array were performed in triplets for young adult and old animals.A total of 6RNA samples of ventricular tissue representing 3individual young adult and 3individual old mice were shipped on dry ice to Exiqon,Inc.,which provided the service for RNA quality verification,microRNA array hybridiza-tion and comprehensive statistical analysis.The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO)database (/geo/)under ac-cession no.GSE32935.Briefly,each pair of young adult and old mouse samples were labeled with Hy3and Hy5fluorescent dyes respectively and hybridized to a miRCURY LNA TM mouse microRNA Array (version 11.0),which held 648mature micro-RNA probes,as well as perfectly matched and mismatched probes for quality control.After signal amplification,the background was subtracted and normalized using LOWESS (Locally Weighted Scatter plot Smoothing)regression algorithm.This within-slide normalization was performed to minimize differences between the colors in an intensity-dependent manner.The array output was received in Excel spreadsheets containing the normalized micro-RNA expression profiles in each heart sample;the expression comparison between old versus young adult heart samples and ‘‘Expression Matrix’’containing normalized Hy3/Hy5ratios (log2transformed)from all hybridizations.The list was sorted based on the most variant expressed miRNAs comparing the two sample types.65miRNAs passed the filtering criteria with an average ‘‘log-Median-Ratio’’.0.58,which represents at least .1.5-fold change in microRNA expression,and the microRNA expression in all three pairs (young adult versus old)was in the same direction.Figure 1.The signal intensity of miRNAs expressed in the mouse heart.There was no significant difference in the overall signal intensity of total microRNAs between young adult (YA)and old hearts.doi:10.1371/journal.pone.0034688.g001Figure 2.The number of miRNA that were differentially expressed in the heart.Among the 599microRNAs that were expressed in the heart,272miRNAs were down-regulated,while 327miRNAs were up-regulated in the old compared with young adult hearts.doi:10.1371/journal.pone.0034688.g002Real-time RT-PCR quantitation of pri-miRNAs and mature miRNAsTo quantitate the expression of the miRNA primary transcripts and the miRNA mature forms,Real-time RT-PCR was performed.To select a proper internal loading control for RT-PCR,we examined the expression of 5S ribosomal RNA (5S RNA)and U6snRNA in young adult versus old hearts.We found that the expression of 5S RNA remained unchanged in young adult vs.old,while U6expression changed significantly in young adult vs.old hearts.Therefore,5S RNA was used as an internal loading control.Detection of pri-miRNAs.The primers for the detection of pri-miRNAs were designed using PRISM Primer Express 3.0software (Applied Biosystems),and synthesized at Integrated DNA Technologies Inc.The first-strand cDNA synthesis was carried outusing random hexamer primer,and the PCR was performed using the following primers:pri-mir-17forward 59-gctttggctttttcctttttg-39,pri-mir-17reverse 59-cctcactgcagtagatgcaca-39;pri-mir-21forward 59-ccagagatgtttgctttgctt-39,pri-mir-21reverse 59-tgccatgagattcaacagtca-39;pri-mir-27a forward 59-tttgatgcc agtcacaaatca-39,pri-mir-27a reverse 59-agccactgtgaacacgacttt-39;pri-mir-93forward 59-cacctcacctaatgaccctca-39,pri-mir-93reverse 59-caagtcctagccctcatggat-39;pri-mir-466d forward 59-cacatgcaa cacacacatatgaa-39and pri-mir-466d reverse 59-ctgattctggcaa gcattttc-39.Detection of mature miRNAs.To detect the mature miRNA strand of either miR or miR*,the first-strand cDNA synthesis was carried out using a universal reverse primer and the Universal RT microRNA PCR system (Exiqon).The RT-PCR reagents and the primers for miR-17,miR-21,mir-21*,miR-27,miR-93,miR-466d-3p and the 5S RNA reference primers that were used as endogenous control were purchased from Exiqon.The PCR amplification was performed in a 7900HT Fast Sequence Detector System (Applied Biosystems)with following program:Cycle 1,95u C for 10minutes.Cycle 2,40cycles of 95u C for 15seconds,60u C for 60seconds.Cycle 3,95u C for 15seconds,60u C for 15seconds,95u C for 15seconds.CT values were automatically obtained.Relative expression values were obtained by normalizing CT values of the miRNA genes in comparison with CT values of the endogenous control (5S RNA)using the CT method [25,26].Transfection AssaysThe Ago1expression plasmid is a gift of Dr.E.K.L.Chan,and the Ago2expression plasmid is a gift of Dr.T.Tuschl.The plasmid construct that contains the mir-21gene was generated by PCR and cloning.Briefly,the DNA fragment containing mir-21gene was amplified using forward primer 59-tagaattc tgcccaggcttt-tatgtattg -39and reverse primer 59-ta ctcgag ggcattgcttttcaagtatgg -39,and then cloned into pCDNA3.1(+)vector.The DNA construct was confirmed by sequencinganalysis.Figure 3.The miRNAs that were increased during adult aging.Thirty-four miRNAs were increased in old (O)vs.young adult (YA)hearts.doi:10.1371/journal.pone.0034688.g003The cardiac muscle cell line H9C2was cultured in DMEM medium containing 10%newborn bovine serum (Invitrogen).Transient transfections were carried out with the Lipofectamine 2000reagents (Invirogen)as previously described [24].At 4h after the transfection was initiated,the H9C2cells were placed in Dulbecco’s modified Eagle’s medium with 10%newborn bovine serum and incubated overnight.The cells were then harvested and the total RNA was isolated using miRNeasy Mini kit (Qiagen)andFigure 4.The miRNAs that were decreased during adult aging.Thirty-one miRNAs were decreased in old (O)vs.young adult (YA)hearts.doi:10.1371/journal.pone.0034688.g004Table 2.The 32miRNAs and their corresponding miRNA gene clusters.Cluster #miRNA cluster miRNAs differentially expressed in O vs YA hearts #1mir-29b-2,29c miR-29c#2mir-466-467-669miR-466a-5p,miR-466b-5p,miR-466c-5p miR-297a*,miR-466d-3p,miR-466d-5p,miR-466f-3p,miR-466i,miR-467b*,miR-467e*,miR-467f,miR-467f,miR-467g,miR-669d,miR-669e,miR-669f#3mir-106b-93-25miR-106a #4mir-29b-1,29a miR-29b#5mir-290,293,295miR-290-5p,miR-291b-5p #6miR-23a ,27a ,24-2miR-27a #7mir-379,411,758miR-380-5p #8mir-23b-27b-24-1miR-27b#9mir-17-19a-92a-1miR-17,miR-19a,miR-19b,miR-20a #10mir-106a-92a-2miR-25,miR-93,#11mir-222,221miR-221,miR-222Total:11miRNA clusters32miRNAsApproximately half of the miRNAs (32out of 65miRNAs)that were differentially expressed in the old heart belong to 11miRNA gene clusters.These 32miRNAs had greater than 1.5-fold change in old (O)versus young adult (YA)hearts.Other miRNAs in the clusters with less than 1.5-fold change in expression are not shown in this table.doi:10.1371/journal.pone.0034688.t002RNase-free DNase I according to the manufacture’s instruction manual.Individual transfection experiments were carried out in triplicate,and the results were reported as average(mean6S.D.) from representative experiments.Statistical analysisData are given as mean values6SD,with n denoting the number of experiments unless otherwise indicated.The differen-tially expressed microRNAs with at least a1.5-fold change were identified using a t-test with a cut off p-value(p,0.05). Results1.Differential expression of cardiac microRNAs in the aging heartIt has been reported that microRNA expression wasaltered in cardiac hypertrophy,congestive heart failure and also in senescent cells[27–31].To test the hypothesis that the expression of microRNA may also change during cardiac aging,we compared microRNA profiles in the young adult(YA,4-month-old)versus old(O,24-month-od)mouse hearts.The Exiqon microRNA array (version11.0)was employed for the microarray analysis.An overview of the total microRNA expression in YA versus old hearts showed that the total microRNA signal intensity was 311248in YA hearts versus310447in old hearts(Figure1);there was no significant difference between the two age groups(NS, n=3).However,the comparison of each individual microRNA expression between old and YA showed that45%of the microRNAs were down-regulated in old versus YA,while55% of the microRNAs were up-regulated in old versus YA(Figure2, p,0,01,n=3).These data indicate that while the expression of many of the individual microRNAs changed in advancing age,the healthy old heart was still able to keep the overall microRNA expression close to that of the YA heart.To examine the miRNAs that were significantly altered during aging,the expression of miRNAs in YA versus old hearts were compared.It was found that the expression level of65miRNAs Figure 6.The miRNA gene clusters and their genomic structures of cluster#5,cluster#8,which are part of11 miRNA gene clusters shown in figure5,6and7.The number of miRNA genes in each cluster ranged from2to71.In10out of11 clusters,the expression of most miRNA genes in that cluster was in the same direction(the arrow q for increased expression,and the arrow Q for decreased expression).Only in one cluster,cluster#7(Figure6C), were most of the miRNA genes not expressed in the same direction. Please note that part of the miRNAs with changes in expression greater than1.5fold in old(O)vs.young adult(YA)hearts were listed in table2. doi:10.1371/journal.pone.0034688.g006Figure 5.The miRNA gene clusters and their genomic structures of cluster#1,cluster#4,which are part of11 miRNA gene clusters shown in figure5,6and7.The number of miRNA genes in each cluster ranged from2to71.In10out of11 clusters,the expression of most miRNA genes in that cluster was in the same direction(the arrow q for increased expression,and the arrow Q for decreased expression).Please note that part of the miRNAs with changes in expression greater than1.5fold in old(O)vs.young adult (YA)hearts were listed in table2.doi:10.1371/journal.pone.0034688.g005changed over1.5-fold in old compared to that of YA hearts. Among them,34miRNAs were up-regulated,while31were down-regulated(Table1and Figure3,4).2.Multiple microRNA clusters are affected in the aging heartThe miRNA genes are distributed across chromosomes either individually,or in clusters.A miRNA cluster is a group of miRNA genes located within a short distance on a chromosome.Based on miRBase database()definition,clustered miRNAs are a group of miRNA genes located within10Kb of distance on the same chromosome.To test the hypothesis that adult aging may affect both individually distributed miRNAs and the microRNAs in clusters,we examine the genomic location of the65miRNAs that were differentially expressed in the aged heart.We found that approximately one half of the differentially expressed miRNAs was individually distributed and the other half was in miRNA clusters.The32miRNAs shown in Table2had1.5-fold change in expression in old compared to that of YA hearts.The miRNA genes that corresponded to the32miRNA mature forms were in 11clusters with their other cluster member(s)(Figure5,6,7and8). Each clusters had at least2genes,with mir-466,467,669cluster (cluster#2)having71genes and mir-379,410cluster(cluster #7)having38genes.Since genes in a miRNA cluster may share a common gene promoter and may be regulated as a whole transcriptional unit,it could be of interest to determine whether any entire cluster changed in the same direction,either up or down.After examination,it was found that the majority of the miRNAs in10of the11clusters(all clusters except cluster#7, Figure6C)tended to change in the same direction as the other members in that cluster(Figure5,6and7),although not all of them had a1.5-fold change(p,0.01)in expression,and not every change was significant(p.0.05).3.Age-related changes in the expression of miRNA guide strands and their corresponding primary transcripts The transcription of pri-miRNA is the first step in miRNA biogenesis[32].Pri-miRNA is first cleaved by the microprocessor complex into pre-miRNA,which is then cleaved by the RNase III enzyme Dicer and become mature microRNAs as a guide strand (miR)and a passenger strand(miR*).During typical miRNA biogenesis,the miR strand is preferentially selected for entry intoa RISC complex,whereas the miR*strand could be degraded [33–35].To test the hypothesis that transcriptional regulation plays an important role in the regulation of the mature microRNA form,we examined pri-miRNA and miR with real-time PCR.As shown in Figure9,the miRNA guide strand miR-17,miR-21, miR-27a and miR-93had an age-related increase accordingly with their corresponding primary transcripts pri-mir-17,pri-mir-21, pri-mir-27and pri-mir-93.Similarly,both mir-466d primary transcript and miR-466d-3p showed an age-related decrease in expression.To further examine whether the expression of both pri-miRNA transcript and the miR strand change during maturation and aging,we compared the pri-mir-21and the miR-21levels in the mouse heart from1month to24months of age.As displayed in Figure10,both miR-21and pri-mir-21levels showed no significant change from1month to6months of age,but started to increase after6months of age.Both miRNA forms reached their highest levels at the age of18months,then decreased afterwards;both curves showed a reversed‘‘V’’shape during adult aging.4.Age-related changes in the expression of miRNA passenger strand(miR*)in the mouse heartIn the cytoplasm,the pre-miRNA becomes cleaved by Dicer, which results in the formation of a22nt double-stranded miRNA duplex,with one being the guide strand(miR)and the other,the passenger strand(miR*).Recent deep-sequencing efforts indicate that a large number of miR*s are not degraded,but rather associate with Ago1or Ago2and remain functional[15,17–20]. An age-related change in the expression of miRNA passenger strands was observed in the present study.Five miR*s:miR-21*, miR-24-2*,miR-138*,miR-297a*,miR-467b*and miR-467e*, were decreased at least1.5-fold,while miR-24-2*was increased at least1.5-fold in old versus YA hearts(Figure11).Interestingly,the fold of change in expression of miR-21*was the highest among all 65miRNAs that were differentially expressed in old versus YA hearts(Figure4and11).The level of miR-21*strand was examined in comparison with miR-21and pri-mir-21transcript from1month to24months of age.As shown in Figure12,the miR-21*expression level didnot Figure7.The miRNA gene clusters and their genomic structures of cluster#7,cluster#11,which are part of11 miRNA gene clusters shown in figure5,6and7.The number of miRNA genes in each cluster ranged from2to71.In10out of11 clusters,the expression of most miRNA genes in that cluster was in the same direction(the arrow q for increased expression,and the arrow Q for decreased expression).Please note that part of the miRNAs with changes in expression greater than1.5fold in old(O)vs.young adult (YA)hearts were listed in table2.doi:10.1371/journal.pone.0034688.g007undergo significant change from 1month to 9months of age,but increased thereafter,and reached the highest level at age 18months.However,its expression level reduced to its lowest level at 24months of age.The miR-21*expression curve during adult aging resembles a reverse ‘‘V’’shape (Figure 12),which is similar to what was observed in the miR-21and pri-mir-21expression curves.These data suggest that the primary transcript expression level affects both miR-21and miR-21*levels.Bioinformatic analysis using MicroCosm Target Scan (/enright-srv/microcosm/htdocs/targets/v5/#)re-vealed that mmu-miR-21*had 879potential target hits,while Mmu-miR-21had 894hits,95%of them are not overlapped.This finding suggest that the passenger strand is not always passive,but at least in some cases,has an important regulatory role in gene expression as well.5.Age-related change in the expression of Ago1and Ago2Argonaute proteins have endonuclease activity directed against messenger RNA strands that are complementary to their bound miRNA fragments.Argonaut proteins are also partially responsi-ble for selection of the guide strand and destruction of the passenger strand.To test the hypothesis that Argonaute expression may change,thereby affecting both miR and miR*expression level during adult aging,we examined the expression of Ago1and Ago2genes during development,maturation and adult aging by real-time RT-PCR.As shown in Figure 13,Ago1expression level was increased from 1to 2months of age and remained at that level from 2months to 11months,then reached the highest level at age of 18months.Afterward,its level was decreased.The Ago2expression stayed at the same level from 1month to 11monthsofFigure 8.The 11miRNA clusters that were affected during adult aging are located on 10chromosomes.Mouse 6Chromosome contains 2clusters (cluster #10‘‘mir-106a-92a-2’’with 6miRNA genes,and #11‘‘mir-222,221’’with 2miRNA genes).doi:10.1371/journal.pone.0034688.g008Figure 9.The expression of miRNAs correlated with that of their corresponding pri-miRNAs.The mature form of miRNAs:miR-17,miR-21,miR-27and miR-93were up-regulated,as were their underlying pri-miRNAs in the old (O)vs.young adult (YA)hearts.The miR-466d-3p was down-regulated,as was also its pri-mir-466d.doi:10.1371/journal.pone.0034688.g009age,increased after 11months of age and reached the highest level at 18months of age.It then was also reduced after 18months.Both the Ago1and Ago2expression levels resembled a reversed ‘‘V’’shape during adult aging from 11months to 20months of age.To test the effect of Ago1and Ago2on the expression of miR-21and miR-21*,the Ago1or Ago2expression plasmid vector was transfected into cells derived from a cardiac muscle cell line,H9C2.As shown in Figure 14and 15,the overexpression of Ago1or Ago2induced the expression of miR-21or miR-21*,respectively.In addition,both Ago1and Ago2synergistically induced miR-21and miR-21*when the mir-21plasmid vector was co-transfected with either.DiscussionThe present study has several major findings.We found that adult aging significantly impacted the expression of 65miRNAs in the heart;among them 55%miRNAs were increased,while 45%miRNAs were decreased in the old versus young adult hearts.Interestingly,over half of these differentially expressed miRNAs belong to 11miRNA clusters,suggesting that these clusters participate in the complex regulation of cardiac gene expression during adult life.We also observed age-related changes in the expression of pri-miRNA transcript and Ago genes,which likely contribute to age-related changes in both miR and miR*expression in the heart.These data enhance our understanding of the complex mechanism(s)underlying the altered expression of miRNA during adult aging.In the present study,half of the miRNAs that are significantly impacted during adult aging belong to 11miRNA clusters,suggesting the importance of understanding the structure and function of these miRNA clusters.A miRNA cluster is a group of miRNA genes located within a short distance on a chromosome.Many of the miRNA clusters may have one core promoter region and transcriptional start site shared by all miRNA genes within that cluster and were ultimately expressed within a single RNA transcript [36].The length of the transcript typically varies from a couple of kb to over 10kb.For instance,the polycistronic transcript of mir-1-1and mir-133a2cluster is 10kb;the mir-1-2and mir-133a1cluster is 6kb [24].It has been reported that each miRNA cluster usually has 2–3miRNA genes,with the majority of clusters having less than 10miRNA genes [37].However,some clusters could have more than 10miRNA genes.For example,Hertel reported that mir-134cluster has more than 50miRNA genes [37].In the present study,we found that the miR-379,329,667,410cluster has 38mir genes,while the miR-466,467,669cluster,being one of the largest miRNA clusters in mouse genome,has 71miRNA genes.Although a number of miRNA clusters have been reported to be associated with pathological and physiological conditions,the overall expression pattern of miRNA clusters during adult aging has not been extensively studied.Since many miRNA genes within a cluster share a common promoter or utilize a common regulatory machinery,examination of their expression pattern andregulatoryFigure 10.The miR-21expression correlated with pri-mir-21levels from 1month to 24months of age.doi:10.1371/journal.pone.0034688.g010Figure 11.Altered expression of miR*during adult aging.Six miR*s that were differentially expressed in old (O)vs young adult (YA)hearts.doi:10.1371/journal.pone.0034688.g011mechanism could enhance our understanding of the role of miRNAs in the process of adult aging.The effects of these altered microRNAs and miRNA clusters on target gene expression during adult aging and senescence warrant further investigation.Exogenous miRNAs can be introduced into live cells or animals;likewise,endogenous miRNAs can also be knocked-down or deleted in cells or animals with various approaches.However,the effects of the various approaches might not be the same [38].It is believed that the length of the 39untranslated regions (39UTRs)of the target messenger RNAs,the cellular proliferative status,and the abundance of target messenger RNA may affect miRNA regulation [39–42].For instance,functional miRNA target sites tend to reside in 39UTRs,therefore the length of the 39UTRs often correlates with density of the miRNA target sites [41,43].Highly proliferating cells usually express more messenger RNAs with shorter 39UTRs,which might escape miRNA targeting,whereas low proliferating cells,including senescent cells,may express miRNAs with longer 39UTRs [39–41].In addition,transfected (exogenous)miRNAs may compete with endogenous miRNAs for the same protein complex,including RISC protein complex that is needed for miRNA regulation.Therefore,genes that are only targeted by endogenous miRNAs but not by the exogenous miRNA may end up relatively increased [44].In addition,the target messenger RNA abundance could potentially dilute microRNA and siRNA activity,thereby reducing the effectiveness of miRNA regulation [45].Nevertheless,miRNA transfection assays have been shown to target various messenger RNAs and intracellular pathways.For example,miR-494down-regulates IGF2BP1and induces cellular senescence in A549cell line [46].In neural stem cells,miR-106b ,25target the insulin/insulin-like growth factor-1(IGF)signaling pathways and promote neuron growth [47].These factors (various approaches of knock-down vs.deletion,cellular proliferative status,length of 39UTR of target,and abundance of messenger RNA target)allFigure 12.The miR-21*passenger strand expression in comparison with miR-21guide strand and pri-mir-21transcript in mouse hearts,from 1month to 24months of age.doi:10.1371/journal.pone.0034688.g012Figure 13.The expression of Argonaute genes.The cardiac expression of Ago1and Ago2in the mouse,from 1month to 24months of age.doi:10.1371/journal.pone.0034688.g013。
断裂蛋白质内含子反式剪接动力学体外反应硕士论文
新型断裂蛋白质内含子体外剪接动力学的研究生物化学与分子生物学， 2010，硕士【摘要】蛋白质内含子(intein)是镶嵌在宿主蛋白质中的插入序列,在宿主蛋白质成熟过程中能够自我剪切下来,同时将两侧的序列—蛋白质外显子(exteins)通过肽键相连接,形成成熟的蛋白质。
蛋白质的这一成熟过程被称为蛋白质顺式剪接(protein cis-splicing)。
断裂蛋白质内含子(split-intein)是在蛋白质内含子内部区域特定位点发生断裂,形成N-端蛋白(IN)和C-端蛋白(Ic),分别由基因组上相距较远的两个开放阅读框(open reading frame, ORF)编码。
在翻译后成熟过程中,IN和Ic相互识别,重建催化活性中心,介导蛋白质反式剪接(protein trans-splicing)。
通过人工改造断裂蛋白质内含子的断裂位点,使之靠近蛋白质内含子的N-末端或C-末端,即形成了新型断裂蛋白质内含子。
断裂蛋白质内含子介导的蛋白质反式剪接技术在蛋白质纯化、基因治疗、环状多肽制备以及大分子复杂蛋白质的生产方面已取得了一些成果。
新型断裂蛋白质内含子在此基础上扩大了断裂蛋白质内含子的应用,提供了在目的蛋白质的N-末端或C-末端标记任意化学基团的潜力。
在进行应用之前需要对这一... 更多还原【Abstract】 Intein is an internal protein sequence in hostprotein precursor. It can autocatalytically self-excise and concomitantly splice together the flanking sequences (N-and C-exteins) with a peptide bond to produce the mature hostprotein. This process is termed as protein cis-splicing. Split-intein is an intein which splits in specific internal site and two parts of the intein (N-intein and C-intein) encoded by two separate open reading frames located apart in the genome, respectively. In the process... 更多还原【关键词】断裂蛋白质内含子；反式剪接；动力学；体外反应；【Key words】split-intein；trans-spiking；kinetics；in vitro reaction；摘要5-7ABSTRACT 7-8第一章绪论11-271.1 引言11-121.2 蛋白质内含子的发现12-131.3 蛋白质内含子的结构和分类13-151.4 蛋白质内含子的剪接机制15-201.5 蛋白质内含子的应用20-241.6 新型断裂蛋白质内含子24-251.7 课题研究的目的、内容和意义25-27第二章实验材料、基本技术和设计方案27-432.1 实验材料和仪器27-352.2 实验基本技术35-422.3 实验设计方案42-43第三章实验过程43-553.1 断裂蛋白质内含子表达质粒的构建43-473.2 前体蛋白质的纯化47-513.3 断裂蛋白质内含子的体外剪接、交叉反应和剪接条件的优化51-533.4 断裂蛋白质内含子的剪接动力学53-55第四章实验结果与分析55-754.1 断裂蛋白质内含子表达质粒的构建55-584.2 前体蛋白质的纯化58-604.3 断裂蛋白质内含子的体外剪接、交叉反应和剪接条件的优化60-694.4 断裂蛋白质内含子的剪接动力学69-75第五章结论75-775.1 结论75-765.2 创新点765.3 展望76-77参考文献。
甲状腺癌与乳腺癌关系研究进展
了 在 母 乳 中 作 为 一 种 营 养 素 外 ，其 在 正 常 或 患 病 的 乳房中尚无已知的作用。乳腺癌 组 织 能 够 吸 收 放 射性碘，而正常的、非泌乳的乳腺组织并不摄取[29]。 上述研究为育龄女性易患乳腺癌提供了可能的 依据。 4 甲状腺癌与乳腺癌的临床特征
有 研 究 表 明 ，乳 腺 癌 与 甲 状 腺 癌 存 在 一 定 的 关 系 ，两者中任一种癌症患者发生另一种癌症的风险 均高于正常人群[3°_32]。乳腺癌 女 性 患 非 恶 性 甲 状 腺 结 节 比 例 高 于 无 乳 房 肿 瘤 女 性 [33]。值 得 注 意 的 是 ，甲状腺乳头状癌、甲状腺滤泡癌和乳腺浸润性 导管癌或导管内癌是甲状腺合并乳腺癌患者的主 要 病 理 类 型 。对 甲 状 腺 癌 患 者 或 乳 腺 癌 患 者 治 疗 后随访应该常规行甲状腺和乳腺的相关检查[34]。 5 小结
甲状腺癌合并乳腺癌的发生原因目前仍未十 分 明 确 。有 研 究 表 明 ，乳 腺 癌 和 甲 状 腺 癌 具 有 一 些 共同的致病因素，如 激 素 、肿瘤基因易感性、肥 胖 、机 体免疫力或者免疫监控能力下降、放疗与化疗等[7]。 2. I 激 素 乳 腺 癌 通 常 由 雌 激 素 和 孕 激 素 受 体 上
参考文献：
[ 1 ] Marcheselli R , Marcheselli L.Cortesi L ,et al. Risk of second pri mary malignancy in breast cancer survivors：a nested populationbased case-control s tu d y [ J ] .J Breast C ancer,2 0 1 5 ,1 8 ( 4 ) ：378385.
雷公藤甲素对局灶节段性肾小球硬化足细胞损伤的调控机制
•研究报告•雷公藤甲素对局灶节段性肾小球硬化足细胞损伤的调控机制王碧娟12,宋李桃'，吕祎琪2,姜雪\李亚妤1r浙江中医药大学附属广兴医院，杭州310007; 2浙江中医药大学，杭州310053)摘要：0的：通过观察雷公藤屮素（T P)对局灶节段性肾小球硬化（F S G S)大鼠及P A N致小鼠足细胞损伤模型中u P A R和T R P C6的表达影响.探讨T P对足细胞的保护作用方法：将48只大鼠随机分为6组，Conlml组、Mmlel组、T P-U^i L T P-M1.)组、T P-H D组和C S A组除Conlml组外，其余各组均通过单侧谓切除联合M静脉重复注射阿霉素方法建立F S G S大鼠模型，药物组给予不同浓度T P或C S A进行治疗，干预8周后处死，检测大鼠24h尿蛋白、血肌酐、尿素氮：H E染色观察肾组织病理变化，电镜观察肾小球足细胞病变。
Western Biol检测肾组织中u P A R和T R P C6蛋白表达培养小鼠肾足细胞M P C5,分为正常组（Control组）、P A N组（Model组）、P A N+T P组（T P组）、P A N+C S A组（C S A组）C C K-8法检测T P对于P A m秀导的足细胞活力的影响，Western Bl&t检测足细胞u P A R,T R P O)蛋H水平的表达结果：M odel组大鼠域蛋A、血肌酐及尿素氮水平较Conlrnl组显著升高(P c O.O I),肾脏病理检查可见小球局灶节段性硬化，小管扩张及足突融合。
Western Blot发现.与Cmilrol组比较.Model组Podorin表达下降，T R P C6及u P A R表达显著升高（R O.Ol )经T P干预后，大鼠床蛋H、血肌酐、尿素氮及肾脏病理显著改善，肾组织丨>ml〇rin表达升高，T R P C6、u P A R表达下降：随着T P浓度升高，足细胞保护作用逐步增强.高浓度T P的作用效果与C S A基本相似细胞实验进一步证实M P C5经P A N作用48h后，Podocin表达显著下降.T R P C6及iiPAR表达明ffi上T|_,T P治疗可有效逆转丨述改变：结论：T P可通过下调T K P C6及u P A R起到保护足细胞.改善F S G S的作Mi关键词：雷公藤甲素；局灶节段性肾小球硬化；足细胞损伤；尿激酶型纤溶酶原激活剂受体；瞬时受体电位 阳离子通道蛋白6基金资助：国家自然科学基金项目（N o.81673913 ),浙江省中医药科学研究基金项目（N〇.2021Z A098)Regulatory m e c h a n i s m of triptolide on podocyte injury infocal segmental glomerulosclerosisWANG Bi-juan1SONG Li-tao',LYU Yi-qi2,JIANG Xue',LI Ya-yu'('G u a n g x i n g Hospital Affiliated to Zhejiang C hinese Medical University, H a n g z h o u 310007, China;"Zhejiang Chinese M edical University, H a n g z h o u 310053, C h i n a )Abstract! Objective: T o observe the effects of triptolide (TP) on the expression of urokinase type plasminogen activator receptor (u P A R) a n d transient receptor potential channel 6 (T R P C6) in focal segmental glomerulosclerosis (F S G S) rats a n d P A N-induced podocyte injury in mice, and to explore the protective effect of T P o n podocytes. M e t h o d s: Forty-eight rats w ere r a n d o m l ydivided into 6 groups: Control group. M o d e l group, T P-L D group. T P-M D group, T P-H D g r o u p a n d C S A group. Except for theControl group, the other groups w e r e all treated b y unilateral n e phrectomy and repeated injection of doxorubicin in the tail vein toestablish F S G S rat models. T h e drug group w a s treated with different concentrations of T P or C S A, a n d they w e r e sacrificed after8 w e e k s of intervention. T h e 24h urine protein, s e r u m creatinine a n d urea nitrogen in rats w e r e deteded. T h e pathological changesof renal tissue w e r e observed by H E staining, a n d the pathological changes of glomerular podocytes w e r e observed by electronmicroscope. W e s t e r n Blot detected the expression of u P A R and T R P C6 protein in kidney tissue. Cultured M P C5 we r e divided intoControl group. P A N group, P A N+T P group, P A N+C S A group. T h e C C K-8 detected the effect of T P o n the viability of podocytesinduced b y P A N. a n d W e s t e r n Blot detected the protein levels of u P A R and T R P C6 in podocytes. Results: T h e 24h urine protein,s e r u m creatinine a n d urea nitrogen levels of the M o d e l gr o u p w e r e significantly higher than the Control gr o u p (P<0.01). T h e renal通信作者：李亚妤，浙江省杭州市体育场路453号浙江中医药大学附属广兴医院肾内科，邮编：310007,电话:I i>'a>.i i3618@ pathological examination s h o w e d focal segmental sclerosis, tubule expansion, epithelial cell shedding, a n d foot Process fusion. Western Blot examination found that c o m p a r e d with the Control group, the expression of P odocin in the M o d e l g r o u p decreased, and the expression of T R P C6a n d u P A R increased significantly (P<0.01). After T P intervention, rat urine protein, s e r u m creatinine, urea nitrogen a n d kidney pathology w e r e significantly improved, Podocin expression in renal tissue increased, a n d T R P C6 and u P A R expression decreased. A s the concentration of T P increases, the protective effect of podocytes gradually increased, a n d the effect of high concentrations of T P w a s basically similar to C S A.Cell experiments further confirmed that M P C5 treated with P A N for 48h. the expression of Podocin decreased significantly, a n d the expression of T R P C6 a n d u P A R increased T P treatment can effectively reverse the a b o v e changes. Conclusion: T P protects podocytes, reduces proteinuria, a n d improves F S G S b y d o w nregulating T R P C6a n d u P A R.K e y w o r d s：Triptolide: Focal segmental glomerulosclerosis (F S G S); P o d o c y t e injury; Urokinase type p l a s m i n o g e n activator receptor (u P A R); Transient receptor potential channel 6 (T R P C6)F u n d i n g：National Natural Science F o u n dation of C h i n a (N o.81673913), Z h e jiang Traditional C h i n e s e M e d i c i n e Science Research Foundation (N〇.2021Z A098)局灶节段性肾小球硬化症（focal segmental glomerulosclerosis，F SG S)是导致终末期肾病（end-stage renal disease, E S R D)的主要病因之一。
MiR-146a-5p对高脂饮食链脲佐菌素诱导的糖尿病肾病大鼠肾组织的保护作用
— 108 —CHINESE JOURNAL OF ANATOMY V ol.44 No.2 2021 解剖学杂志 2021年第44卷第2期MiR-146a-5p 对高脂饮食/链脲佐菌素诱导的糖尿病肾病大鼠肾组织的保护作用*李 莉1 李 松1 曹 萌2 石小娟1 王 雪1 王顺阁1（1 平顶山市第二人民医院内分泌科，平顶山 467000；2 新乡医学院第一附属医院内分泌科，卫辉 453100）摘要 目的：探究miR-146a-5p 对高脂饮食 （HFD ） 和链脲佐菌素 （STZ ） 诱导的糖尿病肾病 （DN ） 大鼠肾的保护作用及其作用机制。
方法：SD 大鼠随机分为正常对照组、模型组、miR-146a-5p 阴性对照组 （mimic 对照组） 和miR-146a-5p mimic 处理组 （mimic 处理组） 。
测量各组大鼠体质量及最后24 h 饮水量和尿量；全自动生化分析仪检测尿液中尿蛋白，血液中血糖、血脂、尿素氮和血清肌酐含量；H-E 染色观察肾小球损伤；TUNEL 染色观察肾组织凋亡，免疫印迹检测肾组织中凋亡标记物半胱氨酸蛋白酶3（caspase 3，cas3），淋巴细胞瘤-2相关蛋白X （Bax ） 和B 淋巴细胞瘤-2（Bcl-2）的表达； 酶联免疫吸附测定（ELISA ）检测外周血中炎症因子肿瘤坏死因子-α （TNF-α），诱导型一氧化氮合酶 （iNOS ）、白细胞介素（IL ）-6（IL-6）和IL-10的含量，免疫印迹检测肾组织中iNOS 和IL-10的蛋白表达。
结果：与正常对照组相比，HFD/STZ 诱导的模型组大鼠体质量明显降低，饮水量、尿量和血液生化指标显著升高；肾小球体积增大，基底膜明显扩张，伴有大量炎性细胞浸润，且细胞凋亡数目明显增多，Bax/Bcl-2比值和cleaved cas3表达显著上调；炎症因子TNF-α、iNOS 、IL-6和IL-10的水平明显上调。
与模型组相比，mimic 处理组大鼠体质量明显增加，饮水量、尿量及血液生化指标显著降低；肾小球体积增大，基底膜明显扩张及炎性细胞的浸润等病理变化明显缓解，细胞凋亡数显著减少，Bax/Bcl-2比值和cleaved cas3表达显著下调；促炎因子TNF-α、iNOS 和IL-6的水平明显降低，抗炎因子IL-10的水平明显升高，mimic 对照组差异无统计学意义。
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
Expression of SNARE proteins in enteroendocrine cell lines and functional role of tetanus toxin-sensitive proteinsin cholecystokinin releaseEric Neèmoz-Gaillard ,Arlette Bosshard ,Romano Regazzi ,Christine Bernard , Jean-Claude Cuber ,Masami Takahashi ,Stefan Catsicas ,Jean-Alain Chayvialle ,Jacques Abello Y*INSERM Uniteè45,Hoêpital Edouard-Herriot,Pavillon Hbis,69437Lyon Ceèdex3,FranceInstitut de Biologie Cellulaire et de Morphologie,Rue du Bugnon9,1005Lausanne,SwitzerlandMitsubishi Kasei Institute of Life Sciences,Machida,Tokyo194,JapanReceived29January1998;revised version received16February1998Abstract In neurons,synaptic vesicle exocytosis involves the formation of a core complex particle including syntaxin-1, synaptosomal-associated protein of25kDa(SNAP-25)and vesicle-associated membrane protein(VAMP)-2/synaptobrevin. The expression of these proteins was investigated in a panel of cell lines,including lines of endocrine and intestinal origin,by Western blotting and/or immunocytochemistry.The three core complex proteins were detected in the enteroendocrine,chole-cystokinin(CCK)-secreting,cell lines STC-1and GLUTag,and in the endocrine non-intestinal cell lines CA-77and HIT-T15.In contrast,SNAP-25and syntaxin-1were undetected in the intestinal non-endocrine cell lines IEC-6,HT-29and Caco-2, whereas a slight expression of VAMP-2was documented in IEC-6and HT-29cells.Co-immunoprecipitation experiments indi-cated that syntaxin-1,SNAP-25and VAMP-2were present in a complex similar to that identified in brain.In the STC-1cell line, treatment of streptolysin-O-permeabilized cells with tetanus toxin(Tetx)selectively cleaved VAMP-2and VAMP-3/cellu-brevin,and simultaneously abolished Ca2+-induced CCK secre-tion(IC SH V12nM).These results show that endocrine cell lines of intestinal origin express syntaxin-1,SNAP-25and VAMP-2, and suggest a key role for a Tetx-sensitive protein(for example VAMP-2and/or VAMP-3)in the CCK secretion by STC-1cells. z1998Federation of European Biochemical Societies.Key words:Syntaxin;Synaptosomal-associated protein of 25kDa;Vesicle-associated membrane protein;Tetanus toxin; Enteroendocrine cell;Cholecystokinin exocytosis1.IntroductionIn neuronal and hormonal cells,release of the signaling substance depends on the precise targeting of transport vesicles to speci¢c membrane compartments within the regu-lated exocytotic pathway.The SNARE hypothesis[1]pro-poses that this speci¢city is achieved through the mutual rec-ognition of vesicle membrane(v-)and target membrane(t-) SNAREs[soluble NSF attachment protein receptors(where NSF=N-ethylmaleimide-sensitive fusion protein)]that assem-ble into stable docking and fusion complexes.According to this hypothesis,recent reports identi¢ed mammalian SNAREs that are implicated in endoplasmic reticulum to Golgi[2,3], and intra-Golgi transport[4].In neurons,syntaxin-1[5,6], SNAP-25[7](synaptosomal-associated protein of25kDa) and synaptobrevin II[8][also known as vesicle-associated membrane protein2(VAMP-2)]are the three SNARE pro-teins that form the core complex involved in synaptic vesicle fusion with plasma membrane[9^11].Recently,these core complex proteins were reported to be expressed in endocrine cells from the adrenal medulla[12,13],anterior pituitary[13^ 15]and pancreas[13,16^20].Independent evidence for the crucial role of these SNAREs in neurotransmitter and hor-mone exocytosis came from studies using clostridial neurotox-ins,tetanus toxin(Tetx)and botulinum toxins A to G[17^24], that selectively cleave syntaxin-1,SNAP-25or VAMP-2 [22,23,25].Insulin release,for instance,was shown to be im-paired by Tetx[17,19,20]and botulinum toxin serotypes A [18,24],B[17],E[18]and F[24]in permeabilized insulinoma cells.The gut epithelium includes a variety of endocrine cells derived,as endocrine pancreas,from the primary endoderm. The fusion machinery involved in peptide secretion from the di¡use endocrine system of the gut has not been identi¢ed. The aim of this work was therefore to determine in the enter-oendocrine cell lines STC-1and GLUTag the presence of syntaxin-1,SNAP-25and VAMP-2,compared with endocrine and non-endocrine cells of di¡erent origin.We here report that STC-1and GLUTag cells express both syntaxin-1, SNAP-25and VAMP-2,and that these proteins form a SNARE complex.Additionally using the intestinal STC-1 cell line that harbors many features of native intestinal chol-ecystokinin(CCK)-producing cells[26,27],we show that treat-ment of permeabilized cells with Tetx results in a marked inhibition of Ca P -induced CCK exocytosis,thus emphasizing a functional role for a tetanus toxin-sensitive SNARE in a model of intestinal endocrine cells.2.Materials and methods2.1.MaterialsMonoclonal antibody against rat syntaxin-1(6D2)was prepared as described[28].Mouse monoclonal antibody against SNAP-25(SMI 81)was from Sternberger Monoclonals Inc.(Baltimore,USA).Poly-clonal antisera to SNAP-25(residues195^206,common to mouse,rat and human)and to rat VAMP-2(residues2^20)were prepared as previously described[7,13].These antibodies cross-reacted with the di¡erent species used in this study,including human material(6D2 and anti-VAMP-2:Y.Shimazaki and M.Takahashi,unpublished;0014-5793/98/$19.00ß1998Federation of European Biochemical Societies.All rights reserved. PII S0014-5793(98)00209-9anti-SNAP-25:S.Catsicas,unpublished).Polyclonal antisera to rat VAMP-1(MC9;residues1^15)and to rat VAMP-3(MC16;residues 1^14)were kindly provided by Dr.P.De Camilli(Yale University, New Haven,USA).Horseradish peroxidase-and FITC-labeled sec-ondary antibodies were obtained from Jackson Laboratories(West Grove,USA)and from Amersham(Les Ulis,France),respectively. Cell culture media,fetal calf serum(FCS)and additives were from Life Technologies(Cergy Pontoise,France).Rat brain extracts and bovine adrenal chroma¤n cell lysates were generously supplied by Dr.A.Osen-Sand(Glaxo Institute for Molecular Biology,Geneva,Swit-zerland)and Dr.M.F.Bader(INSERM U338,Strasbourg,France), respectively.Streptolysin-O(SL-O)was produced and puri¢ed as de-scribed[29].Tetx light chain was generously provided by Dr.U. Weller(University of Mainz,Germany).Protein G-Sepharose,bovine serum albumin(BSA),Triton X-100,sodium orthovanadate,aproti-nin,leupeptin and phenylmethylsulfonyl£uoride(PMSF)were from Sigma(Saint Quentin Fallavier,France).All other reagents were of analytical grade.2.2.Cell lines and culture conditionsThe following cell lines were used in this study:STC-1(mouse small intestine endocrine cell line,[30]),GLUTag(mouse large bowel endocrine cell line[31]),HIT-T15(hamster beta cell line,ATCC CRL 1777),CA-77(rat medullary thyroid carcinoma[32]),IEC-6(rat in-testinal epithelial crypt cell line,[33]),HT-29and Caco-2[human colon adenocarcinoma cell lines(ATCC HTB38and HTB37,respec-tively)].The following cell culture conditions were used:STC-1cells in RPMI with5%FCS;HIT-T15cells in RPMI with10%FCS,32.5 W M glutathione and0.1W M selenous acid;IEC-6cells in DMEM with10%FCS and0.1IU/ml insulin;CA-77in Ham-F-10/DMEM (1:1)with10%FCS;GLUTag,HT-29and Caco-2cells in DMEM with10%FCS.All culture media were supplemented with2mM glutamine and antibiotics(100IU/ml penicillin and50W M strepto-mycin)and cells were grown in a humidi¢ed CO P/air(5:95%)incuba-tor at37³C.2.3.ImmunoprecipitationCells were solubilized in immunoprecipitation bu¡er(50mM HEPES,150mM NaCl,10mM Na R P P O U,10mM EDTA,2mM Na Q VO R,100mM NaF,1%Triton X-100,100units/ml aprotinin, 20W M leupeptin,0.2mg/ml PMSF,pH7.5)for15min on ice.Cell extracts were clari¢ed by centrifugation for15min at14000U g and then incubated with monoclonal SNAP-25antibody preadsorbed to protein G-Sepharose.Beads were subsequently washed twice in im-munoprecipitation bu¡er,resuspended in Laemmli bu¡er and boiled before separation on12%SDS-PAGE.2.4.ImmunoblotsAfter SDS-PAGE,proteins were transferred onto nitrocellulose sheets(Portran Ba85,Schleicher and Schuell,Ecquevilly,France). Membranes were blocked using5%non-fat dry milk in Tris-bu¡ered saline containing0.2%Nonidet P-40,and exposed to the antibodies overnight at4³C in the same bu¡er.After incubation with appropriate secondary antibodies conjugated to horseradish peroxidase,blots were developed using the ECL method(Pierce,Rockford,USA).Protein concentrations were determined by the Bradford method(Bio-Rad, Ivry sur Seine,France).2.5.ImmunocytochemistrySTC-1and GLUTag cells were grown for two days before immu-no£uorescence staining.Cells were washed in PBS and¢xed for10 min in PBS,4%paraformaldehyde.After washing twice in PBS,cells were permeabilized in PBT(PBS containing0.3%Triton X-100and 0.5%BSA)and then incubated with anti-syntaxin-1(6D2,1/200), anti-SNAP-25(SMI81,1/1000)and anti-VAMP-2(1/250)antibodies for1h.Following incubation with FITC-conjugated anti-mouse or anti-rabbit IgG antibody(1/100),immuno£uorescence was visualized with a Leitz£uorescence microscope or with a Zeiss LSM10laser confocal microscope.No staining was observed in cells treated iden-tically but without addition of primary antibodies.2.6.Streptolysin-O permeabilization,neurotoxin treatment andCCK determinationPermeabilization experiments were performed as described previ-ously[18,34]with slight modi¢cations according to[35].Brie£y,STC-1cells were washed once in a Ca P -free Krebs bu¡er and resus-pended in ice-cold KG bu¡er(in mM:140K -glutamate,5NaCl, 7MgSO R,20HEPES,0.4EGTA,pH7.0)containing1IU/ml SL-O. After5min,the cells were washed free of unbound SL-O by centrif-ugation and resuspension in KG bu¡er at4³C.STC-1cells(2.510S cells/60W l¢nal volume)were then incubated at37³C for10min in KG bu¡er supplemented with2.5mM Na P ATP in the presence of the indicated amount of light chain Tetx[21,36,37].The e¤ciency of per-meabilization(more than95%of cells permeabilized)was checked with0.04%Trypan blue.Cells were stimulated by addition of60W l KG bu¡er containing2.5mM Na P ATP,EGTA(10.2mM,¢nal con-centration)and CaCl P to regulate the level of free Ca P [18]and incubated for7min at37³C.The reaction was then quenched by the addition of200W l ice-cold KG bu¡er and cells were sedimented. The amount of CCK released into the supernatant and the remaining immunoreactive material in the pellets were determined by RIA with antiserum39A as previously described[38].The detection limit and ID SH of the assay were0.5and4.0pM,respectively.Results were expressed as a percentage of total cell content[(peptide released in medium U100)/(peptide released in medium+cell content at the end of the incubation)].2.7.Statistical analysisResults were analyzed by one-way analysis of variance(ANOVA) followed by post hoc comparison of Fisher.Di¡erences between two means with a P value60.05were regarded as signi¢cant.All values were expressed as meansþS.E.M.of at least three experiments.3.Results and discussion3.1.Expression of syntaxin-1,SNAP-25and VAMP-2inintestinal endocrine cell linesThe presence of syntaxin-1,SNAP-25and VAMP-2was investigated by immunoblotting in the enteroendocrine cell lines STC-1and GLUTag,and for comparison in the intesti-nal non-endocrine cell lines IEC-6,HT-29and Caco-2,and in the non-intestinal endocrine cell lines HIT-T15and CA-77 (Fig.1A).Syntaxin-1,SNAP-25and VAMP-2were expressed in the intestinal CCK-producing cell lines STC-1and GLU-Tag,as well as in CA-77medullary thyroid carcinoma cells that transcribe the CCK gene[27].The three SNAREs were also detected in the beta cell line HIT-T15(this report and [17,19,20,24]).Surprisingly,a slight expression of VAMP-2 appeared in the non-transformed intestinal epithelial cell line IEC-6,and in the colon adenocarcinoma cell line HT-29but not Caco-2.In contrast,SNAP-25and syntaxin-1expression was not detected in IEC-6,HT-29and Caco-2cells.As ex-pected[5^8,12],the expression of these proteins was detected at a high level in bovine adrenal chroma¤n cells and in rat brain extracts(Fig.1A).The6D2anti-syntaxin-1antibody used in this study recog-nizes both syntaxin-1A and-1B isoforms.These two isoforms were present in the brain(Fig.1B and[1,6]),as well as in neuroendocrine chroma¤n cells(Fig.1B).In contrast,the syntaxin-1A isoform,which shows a faster electrophoretic mobility[1],was found to be the most abundant isoform in the endocrine cell lines STC-1,GLUTag,CA-77and HIT-T15.A similar pattern of syntaxin-1expression has been de-scribed in pancreatic and anterior pituitary endocrine cells [14^16].Subcellular localization of syntaxin-1,SNAP-25and VAMP-2in the enteroendocrine cell lines STC-1(Fig.2) and GLUTag(data not shown)was examined by immunocy-tochemistry and confocal laser microscopy analysis.Essen-tially all STC-1and GLUTag cells were stained with the anti-SNARE protein antibodies.Syntaxin-1(Fig.2A)and SNAP-25(Fig.2B)staining was localized to the peripheryof the cells,while very little cytoplasmic labeling was ob-served.In contrast,confocal laser microscopy showed apatchy intracellular distribution for VAMP-2(Fig.2C).Over-all,these results suggest that syntaxin-1and SNAP-25could act as plasma membrane t-SNAREs in the enteroendocrine cells STC-1and GLUTag,while the cytoplasmic distribution of VAMP-2suggests a v-SNARE status for this plex formation of syntaxin-1,SNAP-25and VAMP-2in intestinal endocrine cell linesDuring neurotransmitter exocytosis,the targeting of synap-tic vesicles to plasma membrane has been proposed to occur through the formation of a stable ternary complex composed of VAMP/synaptobrevin (v-SNARE),syntaxin-1and SNAP-25(t-SNAREs)[1,9].This complex could be isolated from detergent extracts by immunoprecipitation with syntaxin-1or SNAP-25antibodies [1].The formation of this synaptic core complex was also described in adrenal chroma¤n cells [12],PC12pheochromocytoma cells [13]and RINm5F insuli-noma cells [13].To further examine the role of the SNARE proteins present in enteroendocrine cell lines,we investigated the formation of stable ternary complexes containing syntax-in-1,SNAP-25and VAMP-2by immunoprecipitation with the SNAP-25antibody SMI 81(Fig.3).Immunoblotting of syn-taxin-1,SNAP-25and VAMP-2demonstrated that proteins that are part of the SNARE complex in brain (Fig.3and [1,9])could also be detected in the immunoprecipitate from STC-1,GLUTag,HIT-T15and CA-77cells (Fig.3).No non-speci¢c binding was observed when immunoprecipitation wasperformed without SNAP-25antibody (not shown)or with detergent-extracts from Caco-2cells (Fig.3),that do not ex-press any of the three SNARE proteins investigated here (Fig.1).Similar results were obtained when using the anti-SNAP-25polyclonal antiserum for immunoprecipitation (not shown).3.3.Tetanus toxin cleaves VAMP-2and VAMP-3,and inhibitsCa 2+-induced CCK release in streptolysin-O-permeabilized STC-1cellsTetanus toxin is a zinc-dependent protease that cleaves VAMP-2[22,23,25]and VAMP-3/cellubrevin [39]in a highly selective manner.The toxin was shown to inhibit synaptic vesicle exocytosis [22,23]as well as catecholamine [21]and insulin [17,19,20]release from adrenal chroma¤n and insuli-noma cells,respectively.Here,we permeabilized STC-1cells with streptolysin-O to allow Tetx to enter cells.The e¡ects of the Tetx treatment were analyzed on both VAMPs cleavage,and Ca P -induced CCK secretion (Fig.4).Tetx treatment of SL-O-permeabilized cells was found to cause the cleavage of VAMP-2in a dose-dependent manner,but not of syntaxin-1,SNAP-25or VAMP-1,the latter being expressed in STC-1cells (Fig.4,upper panel).The CCK secretion evoked by a high Ca P concentration (4.2þ0.6%vs.2.1þ0.2%of total cell content for 40W M vs.0.1W M free Ca P ,respectively)was inhibited when cells were treated with Tetx (Fig.4,lower panel).These observations argue for the involvement of teta-nus toxin-sensitive protein(s)in CCK exocytosis from enter-oendocrine cells.This protein could be the SNAREproteinFig.2.Immuno£uorescence location of SNARE proteins in the in-testinal endocrine STC-1cells.Immunocytochemistry was done as described in Section 2with (A)anti-syntaxin-1(1/200),(B)anti-SNAP-25(1/1000)and (C)anti-VAMP-2(1/250)antibodies.Primary antibodies were detected with FITC-coupled secondary antibodies and visualized by indirect £uorescence microscopy (A^B),or by la-ser confocal,in phase contrast (C,left panel)and £uorescence (C,right panel),microscopy.Scale bars =10Wm.Fig.1.Immunoblot analysis of SNAREs proteins in cells and tissue of di¡erent origin.Indicated amounts of solubilized extracts were subjected to SDS-PAGE and transferred to nitrocellulose blot.A:SNARE proteins were visualized with monoclonal anti-syntaxin-1(6D2),monoclonal anti-SNAP-25(SMI 81)and polyclonal anti-VAMP-2antibodies.B:Extracts were loaded onto a 6M-urea SDS-polyacrylamide gel [1].After electrophoresis,proteins were transblotted onto nitrocellulose and immunostained with anti-syn-taxin-1(6D2)antibody.Results are representative of three inde-pendent experiments.VAMP-2identi¢ed in STC-1and GLUTag CCK-producing cells,and involved in insulin secretion from pancreatic beta cells [20].However,a functional role of the ubiquitously ex-pressed tetanus toxin-substrate VAMP-3/cellubrevin [39],also present in STC-1cells (Fig.4,upper panel),cannot be ex-cluded.Indeed,this protein,that was shown to participate with VAMP-2in insulin exocytosis from HIT-T15cells [20],was cleaved by Tetx treatment of STC-1cells (Fig.4,upper panel).In addition,the inhibition of CCK secretion correlated with the cleavage of VAMP-2and of VAMP-3,the half-max-imum and maximum concentrations of Tetx being about 12and 50nM for both VAMPs cleavage and inhibition of CCK release.These concentrations were comparable to those re-quired for VAMP-2and VAMP-3cleavage and insulin exo-cytosis inhibition in insulin-secreting cells [17].Whether VAMP-2or VAMP-3,or both VAMPs,are required in the exocytotic process of CCK remains to be elucidated.In conclusion,this study indicates that the three synaptic core complex proteins,syntaxin-1,SNAP-25,and VAMP-2,are expressed in enteroendocrine CCK-secreting cell lines.In addition,a Tetx-sensitive protein (for example,VAMP-2and/or VAMP-3)is required for Ca P -induced CCK exocytosis in the STC-1cell line.A next challenge will be to understand how physiological stimuli,such as nutrients,hormones and neurotransmitters,are able to modulate the exocytotic machi-nery of intestinal CCK-producing cells,and how this organ-ization could be disrupted in states of neuroendocrine secret-ing tumors.Acknowledgements:We are grateful for the gift of the di¡erent cell lines to Drs.A.Leiter (New England Medicine Center,Boston,USA),D.Drucker (Toronto Hospital,Toronto,Canada),J.Philippe (Centre Meèdical Universitaire,Geneva,Switzerland),A.Kervran (INSERM U376,Montpellier,France)and burthe (INSERM U410,Paris,France).The authors thank Drs.P.De Camilli (Yale University,NewHaven,USA)and U.Weller (Johannes-Gutenberg-Universita ët,Mainz,Germany)for the gift of anti-VAMP-1and anti-VAMP-3antibodies,and Tetx light chain,respectively.We also thank K.Sa-doul,A.Osen-Sand and A.M.Chevrier for helpful discussions at various stages of this work.References[1]So ëllner,T.,Whiteheart,S.W.,Brunner,M.,Erdjument-Bromage,H.,Geromanos,S.,Tempst,P.and Rothman,J.E.(1993)Nature 362,318^324.[2]Subramaniam,V.N.,Peter,F.,Philp,R.,Wong,S.H.and Hong,W.(1996)Science 272,1161^1163.[3]Paek,I.,Orci,L.,Ravazzola,M.,Erdjument-Bromage,H.,Am-herdt,M.,Tempst,P.,Sollner,T.H.and Rothman,J.E.(1997)J.Cell Biol.137,1017^1028.[4]Nagahama,M.,Orci,L.,Ravazzola,M.,Amherdt,M.,Lacomis,L.,Tempst,P.,Rothman,J.E.and Sollner,T.H.(1996)J.Cell Biol.133,507^516.[5]Bennett,M.K.,Calakos,N.and Scheller,R.H.(1992)Science257,255^259.[6]Bennett,M.K.,Garcia-Arraraés,J.E.,Elferink,L.A.,Peterson,K.,Fleming,A.M.,Hazuka,C.D.and Scheller,R.H.(1993)Cell 74,863^873.[7]Oyler,G.A.,Higgins,G.A.,Hart,R.A.,Battenberg,E.,Billings-Fig.4.Cleavage of VAMP-2and of VAMP-3,and inhibition of CCK exocytosis,by light chain Tetx in SL-O-permeabilized STC-1cells.Lower panel:E¡ect of Tetx treatment on Ca P -induced CCK release.STC-1cells (2.510S /tube)were treated with SL-O,then ex-posed to 40W M Ca P in the presence of increasing concentrations of Tetx.Total CCK cell content and basal CCK release represented 1175þ147and 25.3þ2.1fmol/tube,respectively.*,P 60.05as com-pared with 40W M Ca P -stimulated CCK release without toxin (n =3).Upper panel:Corresponding representative immunoblot analysis of cell lysates for the presence of syntaxin-1,SNAP-25,VAMP-1,VAMP-2and VAMP-3to evaluate the speci¢city of Tetxcleavage.Fig.3.Immunoprecipitation of SNAP-25-containing protein com-plexes from extracts of various endocrine cell lines.Cells were solu-bilized in Triton X-100solubilization bu¡er for 15min on ice.As indicated,di¡erent amounts of protein extracts were immunoprecipi-tated with the monoclonal anti-SNAP-25antibody (SMI 81),and analyzed by Western blotting with monoclonal anti-syntaxin-1(6D2),monoclonal anti-SNAP-25(SMI 81)and polyclonal anti-VAMP-2antibodies.The positions of SNAREs and of the high chain IgG (IgG H)are indicated (representative of 3separate ex-periments).ley,M.,Bloom,F.E.and Wilson,M.C.(1989)J.Cell Biol.109, 3039^3052.[8]Elferink,L.A.,Trimble,W.S.and Scheller,R.H.(1989)J.Biol.Chem.264,11061^11064.[9]Soëllner,T.,Bennett,M.K.,Whiteheart,S.W.,Scheller,R.H.andRothman,J.E.(1993)Cell75,409^418.[10]Rothman,J.E.(1994)Nature372,55^63.[11]Suëdhof,T.C.(1995)Nature375,645^653.[12]Roth,D.and Burgoyne,R.D.(1994)FEBS Lett.351,207^210.[13]Oho,C.,Seino,S.and Takahashi,M.(1995)Neurosci.Lett.186,208^210.[14]Aguado,F.,Majoè,G.,Ruiz-Montasell,B.,Canals,J.M.,Casa-nova,A.,Marsal,J.and Blasi,J.(1996)Eur.J.Cell Biol.69, 351^359.[15]Jacobsson,G.and Meister,B.(1996)Endocrinology137,5344^5356.[16]Jacobsson,G.,Bean,A.J.,Scheller,R.H.,Juntti-Berggren,L.,Deeney,J.,Berggren,P.O.and Meister,B.(1994)Proc.Natl.A91,12487^12491.[17]Regazzi,R.,Wollheim,C.B.,Lang,J.,Theler,J.M.,Rossetto,O.,Montecucco,C.,Sadoul,K.,Weller,U.,Palmer,M.and Thorens,B.(1995)EMBO J.14,2723^2730.[18]Sadoul,K.,Lang,J.,Montecucco,C.,Weller,U.,Regazzi,R.,Catsicas,S.,Wollheim,C.B.and Halban,P.A.(1995)J.Cell Biol.128,1019^1028.[19]Wheeler,M.B.,Sheu,L.,Ghai,M.,Bouquillon,A.,Grondin,G.,Weller,U.,Beaudoin,A.R.,Bennett,M.K.,Trimble,W.S.and Gaisano,H.Y.(1996)Endocrinology137,1340^1348.[20]Regazzi,R.,Sadoul,K.,Meda,P.,Kelly,R.B.,Halban,P.A.andWollheim,C.B.(1996)EMBO J.15,6951^6959.[21]Ahnert-Hilger,G.,Weller,U.,Dauzenroth,M.E.,Habermann,E.and Gratzl,M.(1989)FEBS Lett.242,245^248.[22]Schiavo,G.,Poulain,B.,Rossetto,O.,Benfenati,F.,Tauc,L.and Montecucco,C.(1992)EMBO J.11,3577^3583.[23]Montecucco,C.and Schiavo,G.(1993)Trends Biochem.Sci.18,324^327.[24]Boyd,R.S.,Duggan,M.J.,Shone,C.C.and Foster,K.A.(1995)J.Biol.Chem.270,18216^18218.[25]Schiavo,G.,Benfenati,F.,Poulain,B.,Rossetto,O.,Polverinode Laureto,P.,DasGupta,B.R.and Montecucco,C.(1992)Na-ture359,832^835.[26]Chang,C.H.,Chey,W.Y.,Sun,Q.,Leiter,A.and Chang,T.A.(1994)Biochim.Biophys.Acta1221,339^347.[27]Cordier-Bussat,M.,Bernard,C.,Haouche,S.,Roche,C.,Abello,J.,Chayvialle,J.A.and Cuber,J.C.(1997)Endocrinology138, 1137^1144.[28]Yoshida,A.,Oho,C.,Omori,A.,Kuwahara,R.,Ito,T.andTakahashi,M.(1992)J.Biol.Chem.267,24925^24928. [29]Bhakdi,S.,Weller,U.,Walev,I.,Martin,E.,Jonas,D.andPalmer,M.(1993)Med.Microbiol.Immunol.182,167^175. [30]Rindi,G.,Grant,S.G.N.,Yiangou,Y.,Ghatei,M.A.,Bloom,S.R.,Bautch,V.L.,Solcia,E.and Polak,J.(1990)Am.J.Pathol.136,1349^1362.[31]Drucker,D.J.,Jin,T.,Asa,S.L.,Young,T.A.and Brubaker,P.L.(1994)Mol.Endocrinol.8,1646^1655.[32]Muszynski,M.,Birnbaum,R.S.and Roos,B.A.(1983)J.Biol.Chem.258,11678^11683.[33]Quaroni,A.and May,R.J.(1980)Methods Cell.Biol.21B,403^427.[34]Gomperts,B.and Tatham,P.E.R.(1988)Methods Enzymol.219,178^189.[35]O'Sullivan,A.J.,Brown,A.M.,Freeman,H.N.M.and Gom-perts,B.D.(1996)Mol.Biol.Cell7,397^408.[36]Weller,U.,Mauler,F.and Habermann,E.(1988)Naunyn-Schmiedeberg's Arch.Pharmacol.338,99^106.[37]Weller,U.,Dauzenroth,M.E.,Meyer zu Heringdorf,D.andHabermann,E.(1989)Eur.J.Biochem.182,649^656.[38]Aucouturier,S.,Bernard,C.,Roche,C.,Philippe,J.,Chayvialle,J.A.and Cuber,J.C.(1994)mun.200,1382^1390.[39]McMahon,H.T.,Ushkryov,Y.A.,Edelmann,L.,Link,E.,Binz,T.,Niemann,H.,Jahn,R.and Suëdhof,T.C.(1993)Nature364, 346^349.。