The Significance of CXCR4 Expression for the Prediction of Lymph Node Metastasis in Breast Cance
CXCR4在原发性肝细胞癌中的表达及意义
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肝细胞癌
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趋化因子受体CXCR4在神经胶质瘤中的表达及其临床意义
趋化因子受体CXCR4在神经胶质瘤中的表达及其临床意义丛璐璐;胡红超;焦保华【摘要】目的观察CXCR4在正常脑组织及各级神经胶质瘤组织中的表达情况,探讨其与胶质瘤的侵袭性、病理级别及血管生成的关系.方法采用免疫组织化学方法测定5例正常脑组织及38例胶质瘤组织中CXCR4、CD34的表达,实时荧光定量PCR法测定CXCR4mRNA的相对表达量.结果 CXCR4在正常脑组织中阴性表达,胶质瘤组织中阳性表达率为63.16%,各病理级别组的阳性表达率及相对表达量差异均有统计学意义(P＜0.05),且均随级别增加而升高;CD34单克隆抗体标记的微血管密度(microvessel density,MVD)在正常脑组织和脑胶质瘤组中的表达均值分别为3.76±0.48和35.25±9.75,在各级别组中的表达均值差异均有统计学意义(P＜0.01)并随级别增加而升高;MVD在CXCR4表达阴性和阳性的胶质瘤组织中表达均值分别为26.37±5.11和40.43±7.88,差异有统计学意义(P＜0.01).结论 CXCR4与胶质瘤的侵袭性及恶性程度有关并可能参与胶质瘤的新生血管生成;MVD可作为判定肿瘤恶性程度和预后的重要指标.【期刊名称】《河北医科大学学报》【年(卷),期】2011(032)007【总页数】5页(P770-773,封2)【关键词】受体,趋化因子;神经胶质瘤;免疫组织化学【作者】丛璐璐;胡红超;焦保华【作者单位】河北医科大学第二医院神经外科,河北,石家庄,050000;河北医科大学第二医院神经外科,河北,石家庄,050000;河北医科大学第二医院神经外科,河北,石家庄,050000【正文语种】中文【中图分类】R739.41神经胶质瘤是颅内最常见的恶性肿瘤，具体发生机制不清，传统的治疗方法效果不佳。
趋化因子受体CXCI4属于G蛋白偶联受体（Gproteincoupled receptor，GPCI）超家族成员，研究表明［1］CXCI4在多种肿瘤中表达，特异性封闭CXCI4可抑制成瘤率。
辅助蛋白
ReviewAccessory proteins of SARS-CoV and othercoronavirusesDing Xiang Liu a ,⇑,To Sing Fung a ,Kelvin Kian-Long Chong a ,Aditi Shukla b ,c ,Rolf Hilgenfeld b ,caSchool of Biological Sciences,Nanyang Technological University,60Nanyang Drive,Singapore 637551,SingaporebInstitute of Biochemistry,Center for Structural and Cell Biology in Medicine,University of Lübeck,Ratzeburger Allee 160,23538Lübeck,Germany cGerman Center for Infection Research (DZIF),University of Lübeck,Germanya r t i c l e i n f o Article history:Received 13February 2014Revised 17June 2014Accepted 23June 2014Available online 1July 2014Keywords:SARS-CoVOther coronaviruses Accessory proteins Structure and functiona b s t r a c tThe huge RNA genome of SARS coronaviruscomprises a number of open reading frames that code for a total of eight accessory proteins.Although none of these are essential for virus replication,some appear to have a role in virus pathogenesis.Notably,some SARS-CoV accessory proteins have been shown to mod-ulate the interferon signaling pathways and the production of pro-inflammatory cytokines.The structural information on these proteins is also limited,with only two (p7a and p9b)having their structures deter-mined by X-ray crystallography.This review makes an attempt to summarize the published knowledge on SARS-CoV accessory proteins,with an emphasis on their involvement in virus–host interaction.The accessory proteins of other coronaviruses are also briefly discussed.This paper forms part of a series of invited articles in Antiviral Research on ‘‘From SARS to MERS:10years of research on highly pathogenic human coronaviruses’’(see Introduction by Hilgenfeld and Peiris (2013)).Ó2014Elsevier B.V.All rights reserved.Contents 1.Introduction ..........................................................................................................972.Accessory proteins of the SARS-CoV .......................................................................................982.1.Orf3a ..........................................................................................................982.2.Orf3b ..........................................................................................................992.3.Orf6..........................................................................................................1002.4.Orf7a .........................................................................................................1012.5.Orf7b .........................................................................................................1022.6.ORF8a and ORF8b ...............................................................................................1022.7.Orf9b .........................................................................................................1033.Accessory proteins of other betacoronaviruses .............................................................................1044.Accessory proteins in Alphacoronavirus ....................................................................................1055.Accessory proteins in Gammacoronavirus ..................................................................................1056.Conclusions..........................................................................................................106Acknowledgements ...................................................................................................106References . (106)1.IntroductionThe severe acute respiratory syndrome coronavirus (SARS-CoV)was identified as the etiological agent of severe acute respiratory syndrome during the 2002–2003outbreak (Peiris et al.,2003).SARS-CoV belongs to the genus Betacoronavirus of the family Coronaviridae (King et al.,2011).Coronaviruses are enveloped viruses with positive-sense,non-segmented,single-stranded RNA genomes,characterized by club-like projections on the virus parti-cles (Masters,2006).The first two thirds of the coronavirus gen-ome encode the replicase genes,which are translated into two large polyproteins,pp1a and pp1ab,that are processed into 15or 16non-structural proteins (nsp)via proteolytic cleavage (Thiel et al.,2003).The remaining one third of the genome contains ORFs/10.1016/j.antiviral.2014.06.0130166-3542/Ó2014Elsevier B.V.All rights reserved.⇑Corresponding author.Tel.:+6563162862;fax:+6567936828.E-mail address:dxliu@.sg (D.X.Liu).for the structural proteins,namely the spike(S),envelope(E), membrane(M),and nucleocapsid(N)proteins.In addition to these genomic elements shared by other coronav-iruses,the SARS-CoV genome also contains eight ORFs coding for accessory proteins,namely ORFs3a,3b,6,7a,7b,8a,8b,and9b (Fig.1).These proteins are specific for SARS-CoV and do not show significant homology to accessory proteins of other coronaviruses, with the exception of the SARS-like coronavirus SL-CoV-WIV1that was recently discovered in bats(Ge et al.,2013)and has the same set of accessory proteins as SARS-CoV.The potential connection between these accessory proteins and the high virulence of SARS-CoV has led to detailed structural and functional studies.Coronavirus accessory proteins have been generally considered to be dispensable for viral replication in vitro(de Haan et al.,2002; Haijema et al.,2004;Yount et al.,2005;Ontiveros et al.,2001;Shen et al.,2003;Hodgson et al.,2006;Casais et al.,2005).However, several accessory proteins have been shown to exhibit functions in virus-host interactions during coronavirus infection in vivo.For example,deletion of ORF2a(NS2),HE,ORF4,and ORF5a in themouse hepatitis virus(MHV)led to a significant attenuation of the virus in its natural host(de Haan et al.,2002).On the other hand,continuous passage of infectious bronchitis virus(IBV)in cell culture results in a mutation in the3b gene coding for a C-termi-nally truncated3b protein.Interestingly,compared with wild-type virus,the mutant virus has a growth advantage and increases virulence in the chicken embryo(Shen et al.,2003).Similarly, recent studies have suggested that SARS-CoV accessory proteins may confer biological advantages to the virus in the natural host, and contribute to the pathogenesis of SARS(Narayanan et al., 2008).Extensive functional studies in cell culture and animal mod-els have shown that SARS-CoV accessory proteins are involved in a wide variety of cellular processes,such as cell proliferation,pro-grammed cell death,activation of stress response pathways and cytokine production,to name just a few.Notably,some coronaviral accessory proteins have also been shown to modulate the inter-feron signaling,which is of paramount importance for host antivi-ral immunity(Frieman et al.,2007;Dedeurwaerder et al.,2014; Zhang et al.,2013;Cruz et al.,2013).As part of a series of invited articles in Antiviral Research on ‘‘From SARS to MERS:10years of research on highly pathogenic human coronaviruses’’(Hilgenfeld and Peiris,2013),this review summerizes our current knowledge on SARS-CoV accessory pro-teins,including an update on structural and functional studies, with a particular emphasis on the involvement of accessory pro-teins in virus-host interactions.In addition,we will briefly summa-rize the current knowledge on accessory proteins of other coronaviruses.2.Accessory proteins of the SARS-CoV2.1.Orf3aProteins3a and3b(p3a and p3b,previously also known as X1 and X2),comprise274and154amino-acid residues,respectively (Marra et al.,2003;Rota et al.,2003).They are encoded by ORF3a (commonly known as ORF3or U274)and ORF3b and both make up the second largest sub-genomic RNA in the SARS-CoV genome(Tan et al.,2006).So far,the structures of the p3a and p3b are still not known(Bartlam et al.,2005)(see Table1).Protein3a was detected in vivo or in vitro when anti-p3a anti-bodies were used in SARS-infected specimens or patients(Yu et al.,2004).Hence,the protein can be used as a diagnostic marker for patients infected with SARS-CoV.In vivo,the presence of p3a was found in autopsy sections from lung and intestinal tissues of SARS patients(Law et al.,2005).Uniquely,frameshift mutations do not necessarily cause protein3a to be non-functional(Tan et al.,2005)and even the virus can deliberately make use of frame-shift mutations to code for3a variants by exploiting hepta-and octa-uridine sites(Wang et al.,2006).It has been shown that SARS-CoV protein3a is a structural com-ponent by using electron microscopy(Ito et al.,2005).Shen et al. supported thisfinding by demonstrating that the protein can be incorporated into virus-like particles(VLPs)using recombinant baculoviruses expressing E,M,and p3a(Shen et al.,2005).How-ever,for the formation of these VLPs,p3a of SARS-CoV is dispensi-ble(Yount et al.,2005).Being efficiently expressed on the cell surface,the protein was easily detected in a majority of SARS patients due to the triggering of a humoral and cellular immune response in these patients(Lu et al.,2009).In light of immunity, p3a can cause the activation of NF-j B and JNK leading to the upregulation of RANTES and IL-8in A549and HEK293T cells (Kanzawa et al.,2006).It has also been demonstrated to up-regu-latefibrinogen mRNA and protein levels in A549cells(Tan et al., 2005).Protein3a has been shown to be present in both the plasma membrane as well as intracellularly(Ito et al.,2005;Oostra et al., 2006;Tan et al.,2004;Yuan et al.,2005).It can also be detected by Western blot in SARS-CoV particles purified by sucrose gradient centrifugation.Yu et ed confocal microscopy and fraction-ation to show that p3a is distributed in a‘‘punctate pattern’’in the cytoplasm while the majority of its expression is concentrated at the Golgi apparatus(Yu et al.,2004).On top of all thesefindings, Tan et al.have shown that this protein can also be transported to the cell surface and enter the cell through endocytosis(Tan et al., 2004).Additionally,through immunoprecipitation,this protein was shown to interact with SARS-CoV M,S,E,and7a proteins (Tan et al.,2004;von Brunn et al.,2007).Being the largest accessory protein(Narayanan et al.,2008),p3a has an extracellular N-terminus and an intracellular C-terminus (Fig.2)Tan et al.,2004;Lu et al.,2006.Another feature of the pro-tein is the presence of a cysteine-rich domain(aa81–160)Lu et al., 2006.The N-terminal region consists of three transmembrane domains(TMDs)(Tan et al.,2004)(Fig.3).Recently,through com-putational modeling,Kruger and Fischer proposed a theoretical model of the protein being an ion channel through the use of its transmembrane domains(Kruger and Fischer,2009).Chien et al. expressed the full-length p3a and reconstituted it into lipid bilay-ers to characterize the ion-channel activity and suggested that either TMD2or3lines the pore of the channel(Chien et al., 2013).The central region comprising residues125–200of p3a has been shown to be bound to the5’UTR of the SARS-CoV genome Table1SARS-CoV ORF3a.SARS-CoV p3aSubcellular localization:punctate pattern in the cytoplasm,Golgi,plasmamembrane and intracellularlyMinor structural proteinNot required for SARS-CoV replicationDomains and/or regions with characterized functionsj C-terminus(aa209–264)induces cell-cycle arrestj The Yxx U motif(aa160–163):internalizing proteins from the plasma membrane into endosomes;also functions in immune evasion j The diacidic motif(aa171–173):ER exportj Full length p3a:interacts with caveolin-1during virus uptake and release j Full length p3a:activates PERK pathway in the UPR and triggers apoptosis j Full length p3a:activates p38kinase to induce cytochrome-c mediated apoptosisj Full length p3a:activates NF-j B and JNK,induces RANTES and IL-8 production98 D.X.Liu et al./Antiviral Research109(2014)97–109(Sharma et al.,2007),characterized by Sharma et al.by using yeast-three-hybrid screen,electrophoretic mobility shift assay, and ultraviolet crosslinking(Sharma et al.,2007).Intriguingly,just the N-terminal portion of p3a is able to evoke a strong humoral immune response(Zhong et al.,2006).The cysteine-rich domain of p3a serves a multitude of functions and some have suggested its role in the p3a ion channel.The p3a is able to interact with the SARS-CoV S protein by formation of disul-fide linkages through its cysteine-rich regions,mainly the CWLCWKC region(aa127–133)(Zeng et al.,2004).Another impor-tant feature is the oligomerization of p3a as it is able to form homodimers and homotetramers(Lu et al.,2006).Through induc-ing point mutations,Lu et al.showed that Cys133is important for oligomerization of the protein and confirmed the formation of dimers and tetramers through FRET(Lu et al.,2006).Utilizing Xenopus oocytes,their results were suggestive of the tetramers being able to form a potassium-permeable ion channel and modu-lating release of SARS-CoV particles(Lu et al.,2006;Shi et al., 2006).Chan et al.further validated that blocking these ion chan-nels suppressed SARS-CoV-mediated apoptosis(Chan et al.,2009).The C-terminal domain is hydrophilic in nature and contains both the Yxx U(where x represents any aa and U is an amino acid with a hydrophobic,bulky side-chain)and the diacidic motifs(ExD, Asp-x-Glu,where x represents any aa).This domain can play a role in G1cell-cycle arrest by depletion of cyclin D3(Yuan et al.,2007). Interestingly,p3a seems to share a similar topology with the M protein(Marra et al.,2003).The Yxx U motif plays a role in inter-nalization of proteins into various intracellular components, including proteins from the plasma membrane into the endosomes (Bonifacino and Traub,2003;Trowbridge et al.,1993).The diacidic motif is required for ER export(Nishimura and Balch,1997).Tan et al.demonstrated that deletion of the C-terminal domain would prevent the protein from being expressed on the cell surface(Tan et al.,2004).Furthermore,they also hypothesized that the Yxx U motif presented in p3a would allow the internalization of the S protein and prevent its presentation on the cell surface(Tan, 2005).This could be involved in evading host immunity systems or even facilitate the assembly of SARS-CoV.p3a can be detected in the supernatant in two different forms from infected Vero E6cells or Caco2cells(Lu et al.,2006;Huang et al.,2006).Huang et al.demonstrated by sucrose-gradient centri-fugation and densitometric scans the presence of a37-kDa form (protein3a-1)and a31-kDa form(protein3a-2).The37-kDa form was detected in fractions with similar densities(1.18–1.20g/ml)to the SARS-CoV particles,as opposed to fractions containing the 31-kDa form(1.13–1.15g/ml)Tan et al.,2005;Yuan et al.,2007, suggesting that the31-kDa form may be present as extracellular membrane structures while the37-kDa form may be assembled into the mature virons.The presence of these two forms is due to the protein3a being able to be modified post-translationally.This protein can be O-glycosylated as demonstrated by Oostra et al. through electrophoretic mobility-shift assays,transforming the unmodified31-kDa form into a33-kDa form(Oostra et al.,2006). p3a resembles the M protein of other coronaviruses,such as MHV,in terms of(1)the subcellular localization pattern of M pro-tein(Tan et al.,2004;Klumperman et al.,1994;Yuan et al.,2005), (2)having an extracellular N-terminus and a C-terminal endodo-main,(3)spanning the membrane three times(Rottier et al.,1984,1986;Braakman and Van Anken,2000),and(4)being consid-ered a structural protein(Ito et al.,2005;Shen et al.,2005).Despite both M and3a proteins being glycosylated,p3a in Ost-7cells is neuraminidase-sensitive but not O-glycosidase sensitive,in contrast to the M protein where it is sensitive to both(Oostra et al.,2006).Recombinant viruses with ORF3a deleted(i.e.,SARS-CoV D3a) showed significant reduction in virus titres after infecting Vero, MA104,and Caco2cells(Yount et al.,2005).Interestingly,Yount et al.demonstrated that deletion of ORF3and ORF6decreased the overall virus titres as compared to just deleting ORF3a alone, suggesting that ORF3a is non-essential(Yount et al.,2005).In con-trast,there is conflicting evidence that ORF3a may be essential, because of the reduction of viral titres in SARS-CoV-infected cells transfected with a siRNA targeting p3a(Åkerström et al.,2007). We propose that this may be due to the subsequent knockdown of p3b as well,resulting in the lower virus production.Using transmission electron microscopy(TEM),Freundt et al. observed in Vero cells that only in the presence of p3a,intracellular vesicles will be formed,as observed in the pathology in SARS patients(Freundt et al.,2010).The presence of Lamp-1through immunofluorescence can also be detected in these vesicles (Freundt et al.,2010),indicating the formation of late endosomes. Expression of p3a is also required for the Golgi fragmentation for its acquisition of a viral envelope.Through yeast-2-hybrid and FRET assays,Padhan et al.have demonstrated caveolin-1binding to p3a,which may play a role in virus uptake and release (Padhan et al.,2007).p3a has also been associated with ER stress through the activation of the PERK pathway but not the IRE-1 and ATF6pathway(Minakshi et al.,2009).Minakshi et al.analyzed this by transfecting Huh7cells with p3a and luciferase-tagged ER stress-related molecules,showing that it can enhance protein fold-ing,but not activate Endoplasmic Reticulum Associated Degrada-tion(ERAD)Minakshi et al.,2009.The long-term triggering of the PERK pathway can also induce virus-related apoptosis through the expression of its downstream mediators of ATF4and CHOP, similar to IBV infection(Liao et al.,2013).Padhan et al.have shown that protein3a can lead to increased activation of the p38MAP kinase pathway and induce the mitochondria to leak cytochromec to induce apoptosis(Padhan et al.,2008).2.2.Orf3bProtein3b(p3b)is translated from ORF3using an IRES(Rota et al.,2003),overlapping the SARS-CoV E gene as well as the ORF3a. Similarly to the SARS-CoV p3a,p3b does not share any homology with other known proteins(Marra et al.,2003).Moreover,p3b is not expressed in SARS-CoV isolates from bats due to the presence of a stop codon(Ren et al.,2006).Anti-p3b antibodies can also be detected in the sera of infected SARS patients(Guo et al.,2004). Yuan et al.have shown using immunofluorescence that the SARS-CoV p3b is localized in the nucleolus of infected Vero E6cells (Yuan et al.,2005)as well as in the mitochondria(Yuan et al.,2006; Freundt et al.,2009).The shuttling behavior may be due to the presence of a nuclear export sequence and determined by a lepto-mycin B-sensitive mechanism.Furthermore,involvement of p3b has also been demonstrated in the induction of necrosis and apop-tosis independent of its subcellular localization(Khan et al.,2006; Yuan et al.,2005).p3b is also able to inhibit the antiviral responsediagram showing the genome organization of the severe acute respiratory syndrome coronavirus(SARS-CoV).The eight accessory proteinsshown as colored boxes.The50leader sequence(black box),open reading frames(ORFs1a,1b)encoding components of the replication/transcription structural genes spike(S),membrane(M),envelope(E),and nucleo-capsid(N)are also indicated(not drawn to scale).by down-regulating type-I interferon(IFN-b)as well as the mito-chondrial antiviral response(Freundt et al.,2009;Spiegel et al., 2005).Usingflow cytometry,Yuan et al.,showed that COS-7cells transfected with p3b are arrested at the G0/G1phase(Yuan et al.,2005).The protein may play a role in immunomodulation through binding to RUNX1b,as seen from yeast-two-hybrid and co-immunoprecipitation by Varshney et al.Varshney et al. (2012).Additionally,p3b can act as an interferon antagonist through inhibition of IRF3(Kopecky-Bromberg et al.,2007)and activation of AP-1through upregulating the expression levels of JNK and ERK(Varshney and Lal,2011)(see Table2).2.3.Orf6Protein6(p6)is detected in lung and ileum tissues of patients and in SARS-CoV-infected Vero E6cells(Geng et al.,2005).The pro-tein is found to associate with cellular membranes and is mainly localized in the ER and Golgi apparatus,either when expressed via a recombinant murine coronavirus or when it is transfected as an EGFP-fusion protein(Geng et al.,2005;Pewe et al.,2005). By sucrose gradient centrifugation and a virus-capture assay, Huang et al.have demonstrated that p6is incorporated into mature virions(Huang et al.,2007).Moreover,when co-expressed with SARS-CoV S,M,and E proteins,p6is incorporated into VLPs. However,p6is not detected in the virions when expressed via a recombinant murine coronavirus(MHV)(Pewe et al.,2005).These results indicate that specific physical interactions between p6and other SARS-CoV structural proteins may be required for recruiting p6into the virus particles(see Table3).Initial reverse genetic studies by Yount et al.have shown that recombinant SARS-CoV with ORF6deleted(rSARS-CoV-D6)repli-cates similarly to wild-type virus in cell cultures as well as in infected BALB/c mice(Yount et al.,2005).However,a later study by Zhao et al.has demonstrated that,at a low MOI(0.01),rSARS-CoV-D6replicates slower and to lower titres in cell cultures (Zhao et al.,2009).Compared with wild-type virus,lower viral titres as well as lower levels of viral RNA and protein were observed for rSARS-CoV-D6at the early stage of infection,but the differences gradually diminished as the infection progressed. Importantly,when transgenic mice expressing the SARS-CoV receptor hACE2were used,rSARS-CoV-D6-infected mice demon-strated a lower morbidity and mortality,with slightly lower virus titres in lung and brain tissue compared with wild-type virus (Zhao et al.,2009).Moreover,when ORF6was introduced into an otherwise sub-lethal strain of MHV,the recombinant virus dis-played enhanced growth in cell cultures and resulted in lethal encephalitis in infected mice(Pewe et al.,2005;Tangudu et al., 2007).Taken together,these results indicate that SARS-CoV p6 enhances viral replication in vitro and in vivo,and it may serve an important role in the pathogenesis during SARS-CoV infection.SARS-CoV protein6is a63-aa polypeptide with an amphipathic N-terminal portion(aa1–40)and a highly polar C-terminal por-tion.An initial study using GFP-fusion protein and digitonin per-meabilization supported an N-endo,C-endo membrane topology (Netland et al.,2007)(Fig.3).Later,it was confirmed that residues 2–37in the N-terminal region form an a-helix and are embedded in the cellular membrane(Zhao et al.,2009).On the other hand,the C-terminal part contains two signal sequences.The sequence YSEL (aa49–52)is known to target proteins for internalization into endosomes,while the acidic tail signals ER export(Fig.2) (Netland et al.,2007).Utilizing the recombinant MHV expressing SARS-CoV p6as mentioned above,Netland et al.demonstrated that the enhance-ment effect of p6is dependent on the N-terminal amphipathic region but not the C-terminal polar region(Netland et al.,2007). Using transmission electron microscopy,Zhou et al.showed that full-length p6or the N-terminal domain modified the cellular membrane,producing perinuclear vesicles that resemble the dou-ble-membrane vesicles(DMVs)known to be associated with coro-navirus replication(Zhou et al.,2010).The ability to modify intracellular membranes may also explain the observation that SARS-CoV p6induces ER stress in transfected cells(Ye et al., 2008).The p6-induced perinuclear vesicles,similar to the virus-induced DMVs,may derive and reorganize the membrane from the ER,thus leading to perturbation of ER function and induction of ER stress(Ye et al.,2008;Knoops et al.,2008).Notably,p6isFig.2.Schematic diagram showing motifs and domains within SARS-CoV accessory proteins p3a and p6.TM,transmembrane domain.Table2100 D.X.Liu et al./Antiviral Research109(2014)97–109yeast two-hybrid and co-immunoprecipitation(Kumar et al., 2007).These results indicate that the N-terminal domain of p6 may play a role in the formation of the replication complex in SARS-CoV-infected cells and serve auxiliary or modulatory func-tions during SARS-CoV genome replication.Protein6was identified as a b-interferon antagonist by Kopecky-Bromberg et al.with the intriguingfinding that overex-pression of p6inhibits nuclear import of STAT1in cells treated with IFN-b(Kopecky-Bromberg et al.,2007).Further studies by the same group revealed that p6can physically interact with kary-opherin alpha2(KPNA2)and tether it to the rough ER(Frieman et al.,2007).The immobilized KPNA2interacts with karyopherin beta1(KPNB1)and prevents it from forming a complex with STAT1to facilitate nuclear import(Zhao et al.,2009).The KPNA2-interacting region in p6is mapped to10aa in the C-terminal region (Frieman et al.,2007).Later,Hussain et al.confirmed that p6but not its C-terminally truncated mutant(p6D C)impeded the nuclear import of proteins harboring a classical nuclear localization signal (NLS)(Hussain et al.,2008).Further studies also demonstrated that the C-terminal tail is necessary but not sufficient to impede nuclear import,and a lipophilic N-terminus context is required(Hussain and Gallagher,2010).Taken together,these results point to a role of the C-terminal domain of p6in modulating host protein nuclear transport and type-I interferon signaling,which may be important for immune evasion during SARS-CoV infection.One recent study has demonstrated a physical interaction and co-localization between SARS-CoV p6and another accessory pro-tein,9b(Calvo et al.,2012).Other studies have also shown that when overexpressed,SARS-CoV p6can stimulate DNA synthesis in cell culture and suppress the expression of co-transfected con-structs(Geng et al.,2005;Hussain et al.,2008;Gunalan et al., 2011).The physiological significances of thesefindings in SARS-CoV replication and pathogenesis are currently unknown.2.4.Orf7aSARS-CoV accessory protein7a(p7a,also known as ORF8,U122, or X4)is detected in SARS-CoV-infected Vero E6cells and in lung specimens from SARS patients(Chen et al.,2005;Fielding et al., 2004;Nelson et al.,2005).The protein is localized in the ER and ER-Golgi intermediate compartment(ERGIC)in SARS-CoV-infected cells and in transfected cells(Fielding et al.,2004;Nelson et al., 2005).Using sucrose gradient centrifugation and a virus capture assay,Huang et al.have shown that SARS-CoV p7a is a structural protein incorporated into mature virions(Huang et al.,2006).Although p7a physically interacts with the SARS-CoV S protein and accessory protein3a,neither S nor p3a is required for recruit-ing p7a into the virus particle;since p7a-containing VLPs can be produced in cells expressing only SARS-CoV M,E,and7a(Tan et al.,2004;Huang et al.,2006)(see Table4).An early study based on reverse genetics has shown that dele-tion of ORF7a from the SARS-CoV genome does not significantly affect viral RNA synthesis and replication efficiency in cell culture (Yount et al.,2005).Schaecher et al.have also found that deletion of both ORF7a and7b does not significantly affect SARS-CoV repli-cation in transformed cells and in infected golden Syrian hamsters (Schaecher et al.,2007;Pekosz et al.,2006).Later,Dediego et al. confirmed that recombinant SARS-CoV with ORF6,7a,7b,8a,8b, and9b deleted produced viral particles with similar morphology as wild-type SARS-CoV and replicated similarly in transgenic mice expressing the SARS-CoV receptor(DeDiego et al.,2008).These results indicate that p7a is not essential for SARS-CoV replication in vitro and in vivo.SARS-CoV p7a is a type-I transmembrane protein with122 amino-acid residues in length(Fig.3).The15N-terminal residues constitute a signal peptide,which is cleaved by signal peptidase in both SARS-CoV-infected cells and in transfected cells(Fielding et al.,2004;Nelson et al.,2005).The aa residues16–96comprise the luminal domain,which folds into a compact seven-stranded b sandwich that resembles members of the Ig superfamily (Nelson et al.,2005).The sequence and structural homology between SARS-CoV p7a and ICAM-1suggested that p7a may inter-act with the lymphocyte function-associated antigen1(LFA-1), which was later confirmed by direct in vitro binding experiments in Jurkat cells(Hänel et al.,2006;Hänel and Willbold,2007).These data suggest LFA-1to be an attachment factor or even receptor for SARS-CoV on human leukocytes,although a majority of p7a is found to remain intracellular in SARS-CoV-infected cells(Nelson et al.,2005;Hänel and Willbold,2007).Residues97–117of SARS-CoV p7a are highly hydrophobic and transverse the cellular membrane.The last5residues of the C-terminal tail(KRKTE)form a typical ER retention motif(KKXX or KXKXX,where X is any amino acid).When this motif is mutated from KRKTE to ERETE,p7a fails to be retro-transported to the ER and accumulates in the Golgi,where it undergoes rapid proteolytic processing(Fielding et al.,2004). Moreover,when the transmembrane domain and the short C-ter-minal tail of SARS-CoV p7a are fused with the cell-surface protein CD4,the chimera protein(CD4/orf7a-TM-tail)is found to be par-tially retained in the Golgi(Nelson et al.,2005).Fielding et al.have demonstrated physical interaction between SARS-CoV p7a and the host protein small glutamine-rich tetratricopeptide repeat-con-taining protein(SGT)(Fielding et al.,2006).Previous studies have shown that SGT interacts with the HIV-1accessory protein vpu,Fig.3.Schematic diagram showing the known topological and structural features of SARS-CoV accessory proteins.N-terminus(N)and C-terminus(C)are indicated. -Helices are represented by blue columns and b-strands are represented by red arrows.The topological or structural features of p3b,p8a,and p8b are not well understood.Ribbon diagrams for p7a and p9b are adapted from Nelson et al.(2005) and Meier et al.,2006,respectively.Table4SARS-CoV ORF7a.SARS-CoV p7aSubcellular localization:ER and ER-Golgi intermediate compartmentMinor structural proteinNot required for SARS-CoV replicationDomains and/or regions with characterized functionsj N-terminal(aa1–15):signal peptidej Luminal domain(aa16–96):resembles ICAM-1-fold,bind to LFA-1j Ser44to Val82:essential for cytoplasmic localization and cell cycle arrest j Transmembrane domain(aa98–116):required for interaction with Bcl-X L and the induction of apoptosisj C-terminal tail(aa118–122):ER retention signalj Entire p7a protein:activates NF-j B and JNK for IL-8and RANTES productionj Entire p7a protein:activates p38and inhibits translation of cellular proteins109(2014)97–109101。
胶质瘤下调CXCR4表达和干细胞侵袭能力、VEGF量分泌分析
中国社区医师2021年第37卷第11期CHINESE COMMUNITY DOCTORS 现阶段，临床普遍认为[1]，和非肿瘤干细胞相比，肿瘤干细胞能够自我更新、多向分化，促进新瘤体的产生，且抗化疗、放疗能力更强[2]。
因此，恶性肿瘤复发的根本原因为肿瘤干细胞作用[3]。
本研究分析了胶质瘤下调CXCR4表达对干细胞侵袭能力、VEGF 分泌量的影响。
资料与方法细胞培养：购买美国菌种保藏中心(ATCC)提供的U87细胞，常规培养，在此过程中将含10%胎牛血清的DMEM 充分利用起来，并获取胶质瘤干细胞球。
方法：①获取U87细胞：将AAGACTGACCAATATTGTACA 选取出来，在此过程中严格依据小干扰RNA 设计原则，将其设定为CXCR4cDNA 的干扰靶点，同时比对，在此过程中采用Blast 软件，将和其他基因同源排除掉。
向载体pSilencerTM 3.1-H1hygro 中插入干扰片，应用FuGENE 转染试剂，向U87细胞转入质粒。
转染后36h 将50ng/mL 潮霉素加入培养基中，筛选将稳定转染的U87细胞获取过来。
运用同样方法向U87细胞中转入pSilencerTM3.1-H1hygroNegativeControl 阴性对照质粒，将稳定转染的U87细胞获取过来，将其设定为阴性对照。
运用Westernblot、间接免疫荧光染色鉴定干扰效果。
②体外侵袭实验：使用孔径为8.0μm 的Transwell 小室对胶质瘤干细胞侵袭能力进行检测，用10μL Matrigel 1:2稀释，在37℃包被微孔膜过夜，将各组胶质瘤干细胞球生长因子及B27祛除，吹散为单细胞悬液前DMEM/F12中进行4h 的孵育，并计数。
将细胞悬液0.2mL 加入上室，将含20%胎牛血清的DMEM 加入下室，在37℃的温度下进行20h 的孵育。
室温下doi:10.3969/j.issn.1007-614x.2021.11.001基金项目潍坊市卫健委科研项目计划WFWSJK-2020-152摘要目的：分析胶质瘤下调CXCR4表达对干细胞侵袭能力、VEGF 分泌量的影响。
CXCL12_CXCR4在肾癌中的研究进展
安徽医药ＡｎｈｕｉＭｅｄｉｃａｌａｎｄＰｈａｒｍａｃｅｕｔｉｃａｌＪｏｕｒｎａｌ２０１０Ｆｅｂ；１４（２）・２２７・ＣＸＣＬｌ２／ＣＸＣＲ４在肾癌中的研究进展郝天春综述，周林玉审校（安徽医科大学附属安徽省立医院泌尿外科，安徽合肥２３０００１）摘要肾癌是泌尿系统常见的恶性肿瘤之一，早期症状不明显，发现时往往已发生了转移。
近年来发现趋化因子ＣＸＣＬｌ２及其受体ＣＸＣＲ４在肾癌转移中发挥着重要的作用，对ＣＸＣＲ４检测有利于判断肾癌转移及患者的预后。
ＣＸＣＲ４靶向治疗已在动物模型及临床前试验中取得了成功，有望成为治疗肾癌新的突破口。
关键词：ＣＸＣＬｌ２；ＣＸＣＲ４；肾癌ＲｅｓｅａｒｃｈｐｒｏｇｒｅｓｓａｂｏｕｔＣＸＣＬｌ２／ＣＸＣＲ４ｉｎｒｅｎａｌｃａｒｃｉｎｏｍａＨＡ０Ｔｉａｎ．ｃｈｕｎ．ＺＨＯＵＬｉｎ．ｙｕ（ＤｅｐａｒｔｍｅｎｔｏｆＵｒｏｌｏｇｙ，ＡｎｈｕｉＰｒｏｖｉｎｃ施ｌＨｏｓｐｉｔａｌＡｆｆｉｌｉａｔｅｄ胁ＡｎｈｕｉＭｅｄｗａｌＵｎｉｖｅｒｓｉｔｙ麒扣ｉ２３０００１，Ｃｈｉｎａ）Ａｂｓｔｒａｃｔ：Ｒｅｎａｌｃａｒｃｉｎｏｍａｉｓｏｎｅｏｆｔｈｅｍａｌｉｇｎａｎｔｔｕｍｏｒｉｎｕｒｉｎａｒｙｓｙｓｔｅｒｍ．Ｉｔｈａｄｔｒａｎｓｆｅｒｒｅｄｗｈｅｎｔｈｅｓｙｍｐｔｏｍｆｏｕｎｄ，ｂｅｃａｕｓｅｔｈｅｅａｒｌｙｉｎｄｉｓｔｉｎｃｔｓｙｍｐｔｏｍ．ＣｈｅｍｏｋｉｎｅＣＸＣＬｌ２ａｎｄｉｔｓｒｅｃｅｐｔｏｒＣＸＣＲ４ｐｌａｙｅｄａｎｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｒｅｎａｌｃａｒｃｉｎｏｍａｍｅｔａｓｔａｓ，ＤｅｔｅｃｔｉｎｇＣＸＣＲ４ｗｏｕｌｄｈｅｌｐｔｏｄｅｔｅｒｍｉｎｅｔｈｅｒｅｎａｌｃａｒｃｉｎｏｍａｍｅｔａｓｔａｓｉｓａｎｄｐｒｏｇｎｏｓｉｓ．ＣＸＣＲ４ｔａｒｇｅｔｔｈｅｒａｐｙｈａｄｂｅｅｎｓｕｃｃｅｄｅｄｉｎａｎｉｍａｌｍｏｄｅｌａｎｄｐｒｅｃｌｉｎｉｃａｌｔｒｉａｌｓ，ｗｈｉｃｈｍｉｓｈｔｐｒｏｖｉｄｅａｎｅｗａｐｐｒｏａｃｈｔｏｒｅｎａｌｃａｒｃｉｎｏｍａｔｈｅｒａｐｙ．Ｋｅｙｗｏｒｄｓ：ＣＸＣＬｌ２；ＣＸＣＲ４；ｒｅｎａｌｃａｒｃｉｎｏｍａ趋化因子是一个可调控细胞运动的超家族。
CXCR4在非小细胞肺癌中的表达及意义
Ｌ ｎ ｅ Ｉ Ｘｉ ｇ， ｔ
（ ｅ ａｔ ｅ ｔｏ ａｄａ—ｈｒ ｉ Ｄｓ ａｅ ， ｅ Ｆｒｔ ｆ ｌ ｔｄ Ｈ ｓ ｉ ， ｈ ｎ ｑｎ ｄｃｌＵ ｉ ｒ ｔ） Ｄ ｐ ｒ ｎ ｆ Ｃ ｒｉ ｔｏａ ｃ ｉ ｓｓ ｔ ｉ ｉ ｉ ｅ ｏｐｔ Ｃ ｏ ｇ ｉ Ｍｅ ｉａ ｎｖ ｓｙ ｍ ｃ ｃ ｅ ｈ ｓＡ ａ １ ０ ｇ ｅｉ
【 摘
要 】目的 ：探讨非小细胞肺癌 （ ｏ ｍ ｌ ｃｌ ｌ ｇｃｎｅ， Ｓ Ｌ Ｎ ｎ ｓ ａｌ ｅｌ ｕ ａｃｒＮ Ｃ Ｃ）中 Ｃ Ｃ类趋化因子受体 ４ （ ｈ ｍ ｋ ｅＣ Ｃ ｍ ｔ ｎ Ｘ Ｃ ｅ ｏｉ Ｘ ｏ ４ Ｃ Ｃ ４ 表达的意义， 其是其表达在局部淋 巴结转移 中的作用 。方法： ｒ 尤 未做治疗的 ４ 例 Ｎ Ｃ Ｃ肿瘤 原发灶 、 中 １ ＳＬ 其
【 ｂｔ ｃ】Ｏ ｊｃｉｅ Ｔ ｘｌｒ ｔｅ ｅｐｅｓ ｎ ｏ Ｘ Ｒ ｎ ｎｎ ｓ ａ ｅ ｕ ｇ ｃｎｅ（ Ｓ Ｌ ）ａ ｄ ｆｒ ｅ ｏｅｐｏｅ ｔ Ａ ｓａｔ ｂｅｔ ： ｏｅｐｏｅ ｈ ｘｒｓｉ ｆＣ Ｃ ４ ｉ ｏ ｍ ｌ ｃｌ ｌｎ ａｃｒＮ Ｃ Ｃ ，ｎ ｕｔ ｒｔ ｘ ｌ ｈ ｒ ｖ ｏ ｌ ｌ ｈ ｒ ｅ
ｍｅ ｎ ｎ ｏ Ｃ Ｒ４， ｓ ｅ ｉｌ ｔ ｅ ｒｌ ｏ Ｃ ａ ｉ ｇ ｆ ＸＣ ｅ ｐ ｃａｌ ｙ ｈ ｏｅ ｆ ＸＣ ４ ｉ ｒ ｇｏ ａ ｌｍｐ ｎ ｄ ｓ ｍｅａ ｔｓｓ ｉ ＮＳ Ｌ ． Ｍ ｅ ｈ ｄ ： ＡＢ Ｒ ｎ ｅｉｎ ｌ ｙ ｈ ｏ ｅ ｔｓ ｉ ｎ ａ Ｃ Ｃ ｔｏ ｓ Ｓ Ｃ
CXCL12、CXCR4与IL-6在特发性膜性肾病中的表达及临床意义
㊃论 著㊃C X C L 12㊁C X C R 4与I L -6在特发性膜性肾病中的表达及临床意义*王思微,牛广华辽宁中医药大学附属医院临床检验中心,辽宁沈阳110032摘 要:目的 研究特发性膜性肾病(I MN )患者趋化因子C X C L 12㊁趋化因子受体C X C R 4㊁白细胞介素(I L )-6的表达情况,探讨其对I MN 的诊断价值㊂方法 选取2018年9月至2019年11月辽宁中医药大学附属医院肾穿刺活检诊断为I MN 的患者105例(I MN 组),根据24h 尿蛋白(24h U T P )水平将其分为低蛋白排泄组(51例)与高蛋白排泄组(54例),并收集同期体检健康者作为对照组(55例)㊂采用酶联免疫吸附试验法检测各组C X C L 12㊁C X C R 4㊁I L -6与抗磷脂酶A 2受体(P L A 2R )抗体水平,比较不同病理分期其水平差异,分析C X -C L 12㊁C X C R 4㊁I L -6与抗P L A 2R 抗体的相关性,以及C X C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体与24h U T P 的相关性,并分析上述指标单独检测及联合检测的诊断效能㊂结果 与对照组相比,I MN 组C X C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体水平增高,差异有统计学意义(P <0.05);高蛋白排泄组C X C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体水平与对照组比较,差异有统计学意义(P <0.05)㊂4种病理分期C X C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体水平比较,差异有统计学意义(P <0.05)㊂C X C L 12㊁C X C R 4与抗P L A 2R 抗体呈正相关(r =0.85㊁0.82,P <0.05);C X C L 12㊁C X C R 4㊁抗P L A 2R 抗体与24h U T P 呈正相关(r =0.85㊁0.83㊁0.85,P <0.05)㊂结论 C X C L 12㊁C X C R 4㊁I L -6与抗P L A 2R 抗体联合检测具有更高的诊断效能,可为I MN 临床诊治提供参考㊂关键词:趋化因子; 白细胞介素-6; 特发性膜性肾病; 诊断D O I :10.3969/j.i s s n .1673-4130.2021.05.001中图法分类号:R 692文章编号:1673-4130(2021)05-0513-05文献标志码:AT h e e x p r e s s i o n a n d c l i n i c a l s i gn i f i c a n c e o f C X C L 12,C X C R 4a n d I L -6i n p a t i e n t s w i t h i d i o p a t h i c m e m b r a n o u s n e p h r o p a t h y*WA N G S i w e i ,N I U G u a n gh u a C l i n i c a l L a b o r a t o r y C e n t e r ,L i a o n i n g U n i v e r s i t y o f T r a d i t i o n a l C h i n e s e M e d i c i n e A f fi l i a t e d H o s p i t a l ,S h e n g y a n g ,L i a o n i n g 110032,C h i n a A b s t r a c t :O b je c t i v e T o s t u d y t h e e x p r e s s i o n of c h e m o k i n e C X C L 12,c h e m o k i n e r e c e p t o r C X C R 4,a n d i n -t e r l e u k i n -6(I L -6)i n p a t i e n t s w i t h i d i o p a t h i c m e m b r a n o u s n e p h r o p a t h y (I MN ).M e t h o d s A t o t a l o f 105p a -t i e n t s d i a g n o s e d w i t h I MN b y k i d n e y b i o p s y i n L i a o n i n g u n i v e r s i t y of t r a d i t i o n a l C h i n e s e m e d i c i n e a f f i l i a t e d h o s p i t a l f r o m S e p t e m b e r 2018t o N o v e m b e r 2019w e r e s e l e c t e d (I MNg r o u p)a n d d i v i d e d i n t o l o w p r o t e i n e x -c r e t i o n g r o u p (51c a s e s )a n d h i g h p r o t e i n e x c r e t i o n g r o u p (54c a s e s )a c c o r d i n g t o 24h u r i n a r y pr o t e i n (24h U T P )l e v e l ,a n d h e a l t h y s u b j e c t s (55c a s e s )w e r e c o l l e c t e d a s c o n t r o l g r o u p .U s i n g e n z ym e -l i n k e d i mm u n o s o r -b e n t a s s a y m e t h o d t o d e t e c t e a c h g r o u p C X C L 12,C X C R 4,I L -6a n d a n t i -p h o s p h o l i p a s e A 2r e c e p t o r a n t i b o d y(a n t i -P L A 2R a n t i b o d y )l e v e l s ,c o m p a r e d i f f e r e n t p a t h o l o g i c a l s t a g i n g i t s l e v e l d i f f e r e n c e ,a n a l y z i n g CX C L 12,C X C R 4,I L -6a n d t h e c o r r e l a t i o n o f a n t i P L A 2R a n t i b o d y a n d C X C L 12,C X C R 4,I L -6,a n t i P L A 2R a n t i b o d ya n d t h e c o r r e l a t i o n o f 24h U T P ,a n d t h e ab o v e i n d e x e s s e p a r a t e l y a n d t h e d i a g n o s i s o f jo i n t d e t e c t i o n e f f i c i e n -c y .R e s u l t s C o m p a r e d w i t h t h e c o n t r o l g r o u p,t h e l e v e l s o f C X C L 12,C X C R 4,I L -6a n d a n t i -P L A 2R a n t i b o d i e s i n I MN g r o u p w e r e i n c r e a s e d ,a n d t h e d i f f e r e n c e w a s s t a t i s t i c a l l y s i g n i f i c a n t (P <0.05).T h e l e v e l s o f C X -C L 12,C X C R 4,I L -6a n d a n t i -P L A 2R a n t i b o d i e s i n t h e h i g h p r o t e i n e x c r e t i o n g r o u p w e r e c o m pa r e d w i t h t h o s e i n t h e c o n t r o l g r o u p ,a n d t h e d i f f e r e n c e s w e r e s t a t i s t i c a l l y s i g n i f i c a n t (P <0.05).T h e r e w e r e s t a t i s t i c a l l y s i g-n i f i c a n t d i f f e r e n c e s i n t h e l e v e l s o f C X C L 12,C X C R 4,I L -6a n d a n t i -P L A 2R a n t i b o d i e s a m o n g t h e 4p a t h o l o gi c a l ㊃315㊃国际检验医学杂志2021年3月第42卷第5期 I n t J L a b M e d ,M a r c h 2021,V o l .42,N o .5*基金项目:国家自然科学基金项目(30772880)㊂ 作者简介:王思微,女,主管技师,主要从事风湿免疫实验室诊断研究㊂ 本文引用格式:王思微,牛广华.C X C L 12㊁C X C R 4与I L -6在特发性膜性肾病中的表达及临床意义[J ].国际检验医学杂志,2021,42(5):513-516.s t a g e s(P<0.05).C X C L12a n d C X C R4w e r e p o s i t i v e l y c o r r e l a t e d w i t h a n t i-P L A2R a n t i b o d y(r=0.85,0.82, P<0.05).C X C L12,C X C R4a n d a n t i-P L A2R a n t i b o d y w e r e p o s i t i v e l y c o r r e l a t e d w i t h24h U T P(r=0.85,0.83,0.85,P<0.05).C o n c l u s i o n T h e c o m b i n e d d e t e c t i o n o f C X C L12,C X C R4,I L-6a n d a n t i-P L A2R a n t i-b o d y h a s h i g h e r d i a g n o s t ic e f f i c a c y,w h i c h c a n p r o v ide r ef e r e n c e f o r t h e c l i n i c a l d i ag n o s i s a n d t r e a t m e n t o f I M N. K e y w o r d s:ch e m o ki n e;i n t e r l e u k i n-6;i d i o p a t h i c m e m b r a n o u s n e p h r o p a t h y;d i a g n o s i s膜性肾病是一种引起成人肾病综合征常见的病理分型,可分为病因不明的特发性膜性肾病(I MN)与由自身免疫性疾病㊁感染㊁肿瘤等原因引起的继发性膜性肾病(S MN)㊂现阶段研究证实I MN发病机制与抗磷脂酶A2受体(P L A2R)抗体水平等有关,抗P L A2R抗体被认为是引发足细胞损伤最重要的自身抗体[1]㊂趋化因子是发挥免疫学作用的一群小分子蛋白,具有稳态与炎性双重的趋化作用㊂趋化因子C X C亚家族的成员C X C L12又名基质细胞衍生因子-1α,其结合到受体C X C R4上发挥独特的生物学效应[2],C X C L12/C X C R4轴被认为广泛作用在肿瘤细胞的迁移与分化㊁促进干细胞归巢㊁并诱导炎性反应与免疫调节等方面㊂本研究旨在检测I MN患者血清C X C L12㊁C X C R4与白细胞介素(I L)-6水平,探究其在疾病的发生与发展中的重要作用,并研究其能否成为I MN新的诊断指标,为I MN疾病诊疗提供新的线索㊂1资料与方法1.1一般资料选取辽宁中医药大学附属医院2018年9月至2019年11月经皮穿刺肾活检明确诊断为I MN的患者共105例(I MN组),除外感染㊁肿瘤㊁自身免疫性疾病等原因引起的S MN,全部入选患者均为初次诊断,未经免疫抑制剂或激素治疗㊂I MN组男性53例㊁女性52例,年龄为19~83岁,平均(55.19ʃ6.73)岁,根据患者的24h尿蛋白定量(24h U T P)将I MN患者分为高蛋白排泄组(24h U T Pȡ1.5g/24h)54例与低蛋白排泄组(24h U T P<1.5 g/24h)51例,所有I MN患者按照E h r e n r e i c h-C h u r g 的病理分期标准分为Ⅰ期27例㊁Ⅱ期42例㊁Ⅲ期19例㊁Ⅳ期17例,并选取55例体检健康者作为对照组,无血清学及尿液的异常,其中男性30例㊁女性25例,年龄为22~78岁,平均(48.23ʃ8.12)岁㊂本研究所有研究对象均签订知情同意书,经本院伦理委员会批准㊂1.2仪器与试剂仪器:美国S p e c t r a M a x i D3多功能酶标仪;日立7600-020全自动生化分析仪㊂试剂: C X C L12酶联免疫吸附试验(E L I S A)试剂盒购自南京森贝咖生物科技有限公司,C X C R4E L I S A试剂盒购自武汉博士德生物科技有限公司;I L-6和抗P L A2R抗体检测E L I S A试剂盒购自德国欧蒙公司; 24h U T P专用试剂盒购自四川迈克公司(免疫比浊法)㊂1.3方法本研究所有对象在清晨空腹采血,在4ħ条件下13000r/m i n离心10m i n,收集血清用E L I S A法对C X C L12㊁C X C R4㊁I L-6及抗P L A2R抗体水平进行检测,所有标本均无黄疸㊁脂血或溶血;患者均空腹留取晨尿,并标记24h尿量㊂所有操作均严格按照说明书进行,E L I S A试剂盒采用五点定标,两个浓度的质控品均在控,且具备符合检测要求的线性条件及其他性能要求㊂1.4统计学处理采用S P S S22.0统计软件进行数据分析,其中符合正态分布的计量资料以xʃs表示,多组间比较采用方差分析,进一步两两比较采用L S D-t检验,两组间比较采用t检验;用P e a r s o n相关分析方法进行相关性分析;分析上述各指标单独检测及不同联合方案诊断I MN的灵敏度㊁特异度㊁阳性预测值㊁阴性预测值及受试者工作特征曲线(R O C曲线)下面积(A U C)㊂P<0.05表示差异有统计学意义㊂2结果2.1各组C X C L12㊁C X C R4㊁I L-6㊁抗P L A2R抗体水平比较与对照组相比,I MN组C X C L12㊁C X C R4㊁I L-6㊁抗P L A2R抗体水平增高,差异有统计学意义(P<0.05),见表1㊂高蛋白排泄组C X C L12㊁C X-C R4㊁I L-6㊁抗P L A2R抗体水平与对照组比较,差异有统计学意义(P<0.05);高蛋白排泄组C X C L12㊁C X C R4㊁I L-6㊁抗P L A2R抗体水平与低蛋白排泄组相比明显增高,差异有统计学意义(P<0.05);低蛋白排泄组C X C R4㊁抗P L A2R抗体水平与对照组相比明显增高,差异有统计学意义(P<0.05),见表2㊂表1I MN组与对照组C X C L12㊁C X C R4㊁I L-6与抗P L A2R抗体水平比较(xʃs)组别n C X C L12(μg/L)C X C R4(μg/L)I L-6(p g/m L)抗P L A2R抗体(R U/m L)对照组552.09ʃ0.591.19ʃ0.3620.93ʃ4.087.64ʃ0.48I MN组1057.53ʃ1.338.72ʃ1.4532.23ʃ3.34145.93ʃ44.06t-3.359-5.664-4.356-7.345P0.0150.0040.0240.003㊃415㊃国际检验医学杂志2021年3月第42卷第5期I n t J L a b M e d,M a r c h2021,V o l.42,N o.52.2 不同病理分期C X C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体水平比较 4种病理分期C X C L 12㊁C X -C R 4㊁I L -6㊁抗P L A 2R 抗体水平比较,差异有统计学意义(P <0.05)㊂进一步两两比较,Ⅱ期患者抗P L A 2R 抗体水平高于Ⅰ期患者;Ⅲ期㊁Ⅳ期患者C X -C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体水平高于Ⅰ期患者;Ⅲ期患者C X C R 4㊁I L -6㊁抗P L A 2R 抗体水平高于Ⅱ期患者;Ⅳ期患者C X C L 12㊁C X C R 4㊁抗P L A 2R 抗体水平高于Ⅱ期患者,Ⅳ期患者C X C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体水平高于Ⅲ期患者,差异均有统计学意义(P <0.05),见表3㊂表2 3组C X C L 12㊁C X C R 4㊁I L -6与抗P L A 2R 抗体水平比较(x ʃs )组别nC X C L 12(μg /L )C X C R 4(μg /L )I L -6(p g /m L )抗P L A 2R 抗体(R U /m L )对照组552.09ʃ0.591.19ʃ0.3620.93ʃ4.087.64ʃ0.48低蛋白排泄组514.23ʃ0.944.52ʃ1.04*24.52ʃ2.8981.92ʃ38.54*高蛋白排泄组5410.64ʃ2.61*#12.69ʃ2.80*#39.52ʃ2.98*#206.39ʃ96.42*#F6.48924.5204.10243.539P0.002<0.0010.016<0.001注:与对照组比较,*P <0.05;与低蛋白排泄组比较,#P <0.05㊂表3 不同病理分期C X C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体水平比较病理分期n C X C L 12(μg /L )C X C R 4(μg /L )I L -6(p g /m L )抗P L A 2R 抗体(R U /m L )Ⅰ期273.58ʃ0.322.78ʃ0.7425.02ʃ2.2562.34ʃ8.74Ⅱ期425.76ʃ1.443.25ʃ2.2429.12ʃ8.4196.36ʃ14.08*Ⅲ期198.57ʃ8.11*12.68ʃ3.10*#45.69ʃ10.73*#205.45ʃ36.77*#Ⅳ期1717.01ʃ3.16*#&27.24ʃ5.15*#&36.37ʃ5.11*&334.64ʃ89.07*#&F7.3612.7417.1561.47P0.0150.004<0.0010.001注:与Ⅰ期比较,*P <0.05;与Ⅱ期比较,#P <0.05;与Ⅲ期比较,&P <0.05㊂2.3 各指标间相关性分析 应用P e a r s o n 相关分析显示,C X C L 12㊁C X C R 4与抗P L A 2R 抗体呈正相关(r =0.85㊁0.82,P <0.05),而I L -6与抗P L A 2R 抗体无相关性(r =0.02,P >0.05);C X C L 12㊁C X C R 4㊁抗P L A 2R 抗体与24h U T P 呈正相关(r =0.85㊁0.83㊁0.85,P <0.05),而I L -6与24h U T P 无相关性(r =0.24,P >0.05),见表4㊂2.4 各指标单项或联合检测诊断I MN 的诊断效能C X C L 12㊁C X C R 4㊁I L -6联合抗P L A 2R 抗体诊断I MN 的灵敏度为90.27%,特异度为95.19%,阳性预测值为91.26%,阴性预测值为94.73%,A U C 为0.937,多指标联合检测A U C 优于单项指标检测(P <0.05)或两项指标联合检测(P <0.05),见表5㊂表4 各指标间相关性分析指标抗P L A 2R 抗体rP 24h U T Pr PC X C L 120.85<0.0010.85<0.001C X C R 40.820.0010.830.003I L -60.020.1320.240.512抗P L A 2R 抗体--0.850.001注:-表示无数据㊂表5 C X C L 12㊁C X C R 4㊁I L -6㊁抗P L A 2R 抗体单项或联合检测诊断I MN 的诊断效能项目A U CP灵敏度(%)特异度(%)阳性预测值(%)阴性预测值(%)抗P L A 2R 抗体0.875<0.00174.1987.6375.2387.51C X C L 120.744<0.00181.8070.7280.1771.25C X C R 40.8540.01678.2075.3780.1475.22I L -60.7330.01876.4166.2275.2968.02抗P L A 2R 抗体+C X C L 120.905<0.00182.2990.5183.1791.26抗P L A 2R 抗体+C X C R 40.9120.00279.2789.3077.1388.26㊃515㊃国际检验医学杂志2021年3月第42卷第5期 I n t J L a b M e d ,M a r c h 2021,V o l .42,N o .5续表5 C X C L12㊁C X C R4㊁I L-6㊁抗P L A2R抗体单项或联合检测诊断I MN的诊断效能项目A U C P灵敏度(%)特异度(%)阳性预测值(%)阴性预测值(%)抗P L A2R抗体+I L-60.8890.00775.1890.3676.0690.15抗P L A2R抗体+C X C L12+C X C R40.9260.00184.3491.5185.0891.34抗P L A2R抗体+C X C L12+C X C R4+I L-60.937<0.00190.2795.1991.2694.733讨论I MN是一种足细胞被破坏的自身免疫性肾病,其基本的病理变化是免疫复合物在肾小球上皮下沉积,激活补体㊁促进攻膜复合物的形成,进一步诱导终末分化的足细胞的凋亡,其中免疫因素可能与其密切相关[3-4]㊂在I MN的发生和发展过程中炎症因素为其重要的致病因素之一,慢性炎症是I MN的主要病理特征之一[5]㊂Z H A N G等[6]构建了膜性肾病㊁免疫㊁炎症相关的三联体网络㊂在一系列炎症物质的研究中,趋化因子被认为是具有较强活性的炎性细胞因子,其与肾脏疾病的关系日益被关注,在I MN患者的肾脏组织采用免疫组化的方法检测到一系列趋化因子的高表达,可能加剧疾病发展[7]㊂C X C L12与其特异性的受体C X C R4结合通过激活通路发挥其特定的生物学功效[8]㊂C X C L12/C X-C R4趋化轴因其对肿瘤细胞存活㊁转移和免疫细胞迁移的影响而备受关注[9]㊂在自身免疫性疾病相关的研究中,C X C L12与非典型趋化因子受体A C K R3结合,A C K R3是调节C X C R4功能的非典型趋化因子受体,在多种癌症类型中过表达㊂C X C L12/C X C R4/ A C K R3轴不仅通过干扰细胞迁移,而且通过调节免疫反应,在众多的自身免疫疾病中调控炎性反应及免疫状态㊂C X C L12与C X C R4特异性结合后通过激活F A K及E R K1/2通路介导I L-6的高表达[10],可能与炎性反应增强有关㊂一些关于肾脏疾病尤其是膜性肾病的研究中,C X C L12/C X C R4轴对足细胞的损伤也日益被关注㊂在肾病方面,阻断C X C L12可以防止小鼠肾小球足细胞的损坏,可能参与糖尿病肾病的发病机制[11]㊂在狼疮性肾炎小鼠中拮抗C X C4R也可改善肾病的严重程度与炎症状态[12]㊂S H A等[13]在体外c5b-9诱导的足细胞损伤模型中,足细胞增殖受到抑制,而C X C L12/C X C R4和磷酸化的S T A T3(p-S T A T3)升高㊂膜性肾病作为一种自身免疫肾病,仍旧依赖于肾组织穿刺活检的诊断方法[14],抗P L A2R 抗体是I MN致病重要的靶抗原,具有较高的灵敏度与特异度[15],但仍存在一定的假阳性,多种检测指标的应用辅助I MN诊断显得尤为重要㊂所以,本研究选取C X C L12㊁C X C R4与I L-6,探讨其成为I MN诊断评估足细胞损伤的新指标,同时探究联合检测能否替代对患者有创性的肾穿刺活检病理检测手段,并联合特异性的抗P L A2R抗体,探讨新型诊断模式㊂本研究结果显示,C X C L12㊁C X C R4㊁抗P L A2R抗体与24h U T P呈正相关,可能与足细胞损伤肾小球机械屏障受损有关,符合I MN部分患者存在肾病综合征的临床表现㊂本研究还发现,4种指标联合检测具有更高的诊断效能㊂但是本研究仍存在样本量少㊁入选病例存在偏倚㊁机制研究不足㊁统计学方法单一等问题,在后续研究中应进一步完善㊂参考文献[1]C A T T R A N D C,B R E N C H L E Y P E.M e m b r a n o u s n e-p h r o p a t h y:I n t e g r a t i n g b a s i c s c i e n c e i n t o i m p r o v e d c l i n i c a l m a n a g e m e n t[J].K i d n e y I n t,2017,91(3):566-574.[2]P A V L A S O V A G,B O R S K M,S E D A V,e t a l.I b r u t i n i bi n h i b i t s C D20u p-r e g u l a t i o n o n C L L B c e l l s m e d i a t e d b y t h e C X C R4/S D F-1a x i s[J].B l o o d,2016,128(12):1609-1613.[3]S A N D O R D G,B E C K L H.T h e r o l e o f c o m p l e m e n t i nm e m b r a n o u s n e p h r o p a t h y[J].S e m i n N e p h r o l,2013,33(6):531-542.[4]F R A N C I S J M,B E C K L H,S A L A N T D J.M e m b r a n o u s n e p h r o p a t h y:a j o u r n e y f r o m b e n c h 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[6]Z HA N G C W,L E N G L,L I Z Z,e t a l.I d e n t i f i c a t i o n o f b i-o m a r k e r s a n d d r u g r e p u r p o s i n g c a n d i d a t e s b a s e d o n a n i mm u n e,-i n f l a mm a t i o n-a n d m e m b r a n o u s g l o m e r u l o n e-p h r i t i s-a s s o c i a t e d t r i p l e t s n e t w o r k f o r m e m b r a n o u s g l o-m e r u l o n e p h r i t i s[J].B M C M e d G e n o m i c s,2020,13(1):5.[7]M E Z Z A N O S A,D R O G U E T T M A,B U R G U S M E,e ta l.O v e r e x p r e s s i o n o f c h e m o k i n e s,f ib r o g e n ic c y t o k i n e s, a nd m y o f i b r o b l a s t s i n h u m a n me m b r a n o u s n e p h r o p a t h y[J].K i d n e y I n t,2000,57(1):147-158.[8]J A N S S E N S R,S T R U Y F S,P R O O S T P.T h e u n i q u e s t r u c-t u r a l a n d f u n c t i o n a l f e a t u r e s o f C X C L12[J].C e l l M o l I m-m u n o l,2018,15(4):299-311.[9]S HA H K,P A T E L S,M I R Z A S,e t a l.U n r a v e l l i n g t h e l i n k b e t w e e n e m b r y o g e n e s i s a n d c a n c e r m e t a s t a s i s[J].G e n e,2018,642(1):447-452.[10]P E N G H,K O L B R,K E N N E D Y J E,e t a l.D i f f e r e n t i a le x p r e s s i o n of C X C L12a n d C X C R4d u r i ngh u m a n f e t a ln e u r a l p r o g e n i t o r c e l l d i f f e r e n t i a t i o n[J].J N e u r o i mm u n e P h a r m a c o l,2007,2(3):251-258.(下转第520页)㊃615㊃国际检验医学杂志2021年3月第42卷第5期I n t J L a b M e d,M a r c h2021,V o l.42,N o.5晚期N O具有明显的神经毒性作用,导致严重的神经功能缺损㊂M D A是脂质氧化的最终产物,其水平变化可间接反映组织中氧自由基的变化,S O D作为主要的清除氧自由基的酶,对抗氧自由基对细胞损伤有重要作用[8-9]㊂本研究中大鼠在造模后,氧化应激指标N O和M D A水平明显升高,S O D水平明显降低,说明造模引起的缺血再灌注损伤激活了脑组织的氧自由基,氧化与抗氧化平衡破坏,川芎嗪治疗后氧化指标显著下降,抗氧化指标明显提高,提示川芎嗪可有效调节损伤后脑组织的氧化平衡状态,进而降低氧自由基带来的损伤作用,保护神经细胞㊂在中枢神经系统中,C a2+参与神经细胞兴奋㊁神经递质释放等重要功能㊂钙超载被认为是各种有害因素导致神经元变性的最后共同通道,可通过激活氧化应激反应㊁损伤线粒体等途径引起神经细胞死亡㊂正常神经细胞内C a2+与细胞外相近,主要通过电压依赖型钙通道㊁C a2+-A T P酶等维持C a2+梯度㊂缺血时,细胞能量代谢发生障碍,导致细胞膜C a2+-A T P 酶活性下降,细胞C a2+内流增加,外排减少,从而引起钙超载[10]㊂本研究中川芎嗪可保护线粒体,提高C a2+-A T P酶活性,促进C a2+排出,降低钙超载状态,保护神经组织㊂缺血再灌注损伤发生后,大量白细胞浸润脑组织,产生炎症因子进而加重了脑损伤,已有大量实验证明I L-1㊁I L-6㊁I L-8及T N F-α等细胞因子参与炎性反应[11],但有关川芎嗪对炎症因子调节的报道较少见㊂本研究结果表明M C A O模型建立后大鼠脑组织中炎性反应活跃,各类炎症因子表达明显增加,继而造成神经细胞的损伤,川芎嗪各项炎症因子基因相对表达水平均显著下降,说明川芎嗪可通过抑制炎症发生改善脑组织微环境,促进神经细胞恢复,进一步探究临床川芎嗪治疗脑卒中的作用机制㊂参考文献[1]Y A N G G,Q I A N C,WA N G N,e t a l.T e t r a m e t h y l p y r a z i n ep r o t e c t s a g a i n s t o x y g e n-g l u c o s e d e p r i v a t i o n-i n d u c e d b r a i n m i c r o v a s c u l a r e n d o t h e l i a l c e l l s i n j u r y v i a R h o/R h o-k i n a s es i g n a l i n g p a t h w a y[J].C e l l M o l N e u r o b i o l,2017,37(4): 619-633.[2]Z H O N G M,M A W,Z H A N G X,e t a l.T e t r a m e t h y l p y r a z i n e p r o t e c t s h i p p o c a m p a l n e u r o n s a g a i n s t a n o x i a/r e o x y g e n a t i o n i n j u r y t h r o u g h i n h i b i t i n g a p o p t o s i s m e d i a t e d b y J N K/M A R K s i g n a l p a t h w a y[J].M e d S c i M o n i t,2016,22(1):5082-5090.[3]C H E N J,L I Y,WA N G L,e t a l.T h e r a p e u t i c b e n e f i t o f i n-t r a v e n o u s a d m i n i s t r a t i o n o f b o n e m a r r o w s t r o m a l c e l l s a f-t e r c e r e b r a l i s c h e m i a i n r a t s[J].S t r o k e,2001,32(4): 1005-1011.[4]K OH S H,P A R K H H.N e u r o g e n e s i s i n S t r o k e R e c o v e r y[J].T r a n s l S t r o k e R e s,2017,8(1):3-13.[5]W A N G S,X I A B,Q I A O Z,e t a l.T e t r a m e t h y l p y r a z i n e a t t e n-u a t e d b u p i v a c a i n e-i n d u c e d n e u r o t o x i c i t y i n S H-S Y5Y c e l l s t h r o u g h r e g u l a t i n g a p o p t o s i s,a u t o p h a g y a n d o x i d a t i v e d a m a g e [J].D r u g D e s D e v e l T h e r,2019,13(1):1187-1196. 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靶向CXCR4 PETCT 分子探针在肿瘤诊疗中的应用进展
国际医学放射学杂志ＩｎｔＪＭｅｄＲａｄｉｏｌ２０21Sep 鸦44穴5雪：584-588靶向CXCR4PET/CT 分子探针在肿瘤诊疗中的应用进展孙芳芳杜雪梅*【摘要】趋化因子受体4（CXCR4）在许多肿瘤细胞表面高表达，它可以作为肿瘤干细胞的标志物，其表达与肿瘤的恶性程度及病人的预后直接相关。
靶向CXCR4PET/CT 显像在疾病的诊断、CXCR4靶向治疗病例选择、疗效评价等方面具有很大的应用潜力。
就靶向CXCR4PET/CT 分子探针及其在肿瘤诊疗中的应用研究进展进行综述，总结分析各种探针的特点及临床转化情况，为CXCR4临床应用转化提供帮助。
【关键词】趋化因子受体4；分子探针；正电子发射体层成像；肿瘤中图分类号：R73；R445.3；R445.6文献标志码：AProgress of CXCR4targeted PET/CT molecular probe and its application in tumor SUN Fangfang,DUXuemei.Department of Nuclear Medicine,First Affiliate Hospital of Dalian Medical University,Dalian 116022,China.Corresponding author:DU Xuemei,E-mail:***************【Abstract 】Chemokine receptor 4(CXCR4),highly expressed on the surfaces of various tumor cells,can be used as amarker of tumor stem cells.The expression of CXCR4correlates with the tumor malignancy and the prognosis of patients.CXCR4as a target for PET/CT imaging shows great potentials in the disease diagnosis,case selection for CXCR4-targettreatment and the evaluation of therapeutic effect.We reviewed the research progresses of targeted CXCR4PET/CT molecular probe and its application in tumor diagnosis and treatment,summarized the characteristics and clinicaltransformation of various probes,so as to provide help for the clinical application and transformation of CXCR4.【Keywords 】Chemokine receptor 4;Molecular probe;Positron emission tomography;TumorＩｎｔＪＭｅｄＲａｄｉｏｌ，２０21，44（5）：584-588作者单位：大连医科大学附属第一医院核医学科，大连116022通信作者：杜雪梅，E-mail ：****************审校者DOI:10.19300/j.2021.Z18821肿瘤细胞表面表达多种特异性的受体蛋白，趋化因子受体4（chemokine receptor 4，CXCR4）作为趋化因子受体家族中的一员在多种肿瘤细胞表面高表达，其表达高低与肿瘤的恶性程度及病人的预后直接相关[1-3]。