VHCF-6论文集AM17
Histone Methylation by PRC2 Is Inhibited by Active Chromatin Marks

Molecular CellArticleHistone Methylation by PRC2Is Inhibitedby Active Chromatin MarksFrank W.Schmitges,1,6Archana B.Prusty,2,6Mahamadou Faty,1Alexandra Stu¨tzer,3Gondichatnahalli M.Lingaraju,1 Jonathan Aiwazian,1Ragna Sack,1Daniel Hess,1Ling Li,4Shaolian Zhou,4Richard D.Bunker,1Urs Wirth,5Tewis Bouwmeester,5Andreas Bauer,5Nga Ly-Hartig,2Kehao Zhao,4Homan Chan,4Justin Gu,4Heinz Gut,1 Wolfgang Fischle,3Ju¨rg Mu¨ller,2,7,*and Nicolas H.Thoma¨1,*1Friedrich Miescher Institute for Biomedical Research,Maulbeerstrasse66,CH-4058Basel,Switzerland2Genome Biology Unit,EMBL Heidelberg,Meyerhofstrasse1,D-69117Heidelberg,Germany3Laboratory of Chromatin Biochemistry,Max Planck Institute for Biophysical Chemistry,Am Fassberg11,D-37077Go¨ttingen,Germany4China Novartis Institutes for Biomedical Research,Lane898Halei Road,Zhangjiang,Shanghai,China5Novartis Institutes for Biomedical Research,CH-4002Basel,Switzerland6These authors contributed equally to this work7Present address:Max Planck Institute of Biochemistry,Am Klopferspitz18,D-82152Martinsried,Germany*Correspondence:muellerj@biochem.mpg.de(J.M.),nicolas.thoma@fmi.ch(N.H.T.)DOI10.1016/j.molcel.2011.03.025SUMMARYThe Polycomb repressive complex2(PRC2)confers transcriptional repression through histone H3lysine 27trimethylation(H3K27me3).Here,we examined how PRC2is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2Nurf55-Su(z)12 submodule.Binding of H3is lost if lysine4in H3is trimethylated.Wefind that H3K4me3inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12C terminus.In addition to H3K4me3,PRC2 is inhibited by H3K36me2/3(i.e.,both H3K36me2 and H3K36me3).Direct PRC2inhibition by H3K4me3and H3K36me2/3active marks is con-served in humans,mouse,andfly,rendering tran-scriptionally active chromatin refractory to PRC2 H3K27trimethylation.While inhibition is present in plant PRC2,it can be modulated through exchange of the Su(z)12subunit.Inhibition by active chromatin marks,coupled to stimulation by transcriptionally repressive H3K27me3,enables PRC2to autono-mously template repressive H3K27me3without over-writing active chromatin domains.INTRODUCTIONPolycomb(PcG)and trithorax group(trxG)proteins form distinct multiprotein complexes that modify chromatin.These com-plexes are conserved in animals and plants and are required to maintain spatially restricted transcription of HOX and other cell fate determination genes(Henderson and Dean,2004; Pietersen and van Lohuizen,2008;Schuettengruber et al., 2007;Schwartz and Pirrotta,2007).PcG proteins act to repress their target genes while trxG protein complexes are required to keep the same genes active in cells where they must be expressed.Among the PcG protein complexes,Polycomb repressive complex2(PRC2)is a histone methyl-transferase(HMTase) that methylates Lys27of H3(H3K27)(Cao et al.,2002;Czermin et al.,2002;Kuzmichev et al.,2004;Mu¨ller et al.,2002).High levels of H3K27trimethylation(H3K27me3)in the coding region generally correlate with transcription repression(Cao et al., 2008;Nekrasov et al.,2007;Sarma et al.,2008).PRC2contains four core subunits:Enhancer of zeste[E(z),EZH2in mammals], Suppressor of zeste12[Su(z)12,SUZ12in mammals],Extra-sex combs[ESC,EED in mammals]and Nurf55[Rbbp4/ RbAp48and Rbbp7/RbAp46in mammals](reviewed in Schuet-tengruber et al.,2007;Wu et al.,2009).E(z)is the catalytic subunit;it requires Nurf55and Su(z)12for nucleosome associa-tion,whereas ESC is required to boost the catalytic activity of E(z)(Nekrasov et al.,2005).Recent studies reported that ESC binds to H3K27me3and that this interaction stimulates the HMTase activity of the complex(Hansen et al.,2008;Margueron et al.,2009;Xu et al.,2010).The observation that PRC2is able to bind to the same modification that it deposits led to a model for propagation of H3K27me3during replication.In this model, recognition of H3K27me3on previously modified nucleosomes promotes methylation of neighboring nucleosomes that contain newly incorporated unmodified histone H3(Hansen et al.,2008; Margueron et al.,2009).However,it is unclear how such a positive feedback loop ensures that H3K27trimethylation remains localized to repressed target genes and does not invade the chromatin of nearby active genes.In organisms ranging from yeast to humans,chromatin of actively transcribed genes is marked by H3K4me3, H3K36me2,and H3K36me3modifications:while H3K4me3is tightly localized at and immediately downstream of the transcrip-tion start site,H3K36me2peaks adjacently in the50coding region and H3K36me3is specifically enriched in the30coding region(Bell et al.,2008;Santos-Rosa et al.,2002).Among the trxG proteins that keep PcG target genes active are the HMTases Trx and Ash1,which methylate H3K4and H3K36, respectively(Milne et al.,2002;Nakamura et al.,2002;Tanaka330Molecular Cell42,330–341,May6,2011ª2011Elsevier Inc.et al.,2007).Studies in Drosophila showed that Trx and Ash1 play a critical role in antagonizing H3K27trimethylation by PRC2,suggesting a crosstalk between repressive and activating marks(Papp and Mu¨ller,2006;Srinivasan et al.,2008).In this study we investigated how PRC2activity is modulated by chromatin marks typically associated with active transcrip-tion.We found that the Nurf55WD40propeller binds the N terminus of unmodified histone H3and that H3K4me3 prevents this binding.In the context of the tetrameric PRC2 complex,wefind that H3K4me3and H3K36me2/3(i.e.,both H3-K36me2and H3-K36me3)inhibit histone methylation by PRC2in vitro.Dissection of this process by usingfly,human, and plant PRC2complexes suggests that the Su(z)12subunit is important for mediating this inhibition.PRC2thus not only contains the enzymatic activity for H3K27methylation and a recognition site for binding to this modification,but it also harbors a control module that triggers inhibition of this activity to prevent deposition of H3K27trimethylation on transcription-ally active genes.PRC2can thus integrate information provided by pre-existing histone modifications to accurately tune its enzymatic activity within a particular chromatin context.RESULTSStructure of Nurf55Bound to the N Terminusof Histone H3Previous studies reported that Nurf55alone is able to bind to histone H3(Beisel et al.,2002;Hansen et al.,2008;Song et al., 2008;Wysocka et al.,2006)but not to a GST-H3fusion protein (Verreault et al.,1998).By usingfluorescence polarization(FP) measurements,we found that Nurf55binds the very N terminus of unmodified histone H3encompassing residues1–15(H31–15) with a K D of$0.8±0.1m M but does not bind to a histone H319–38 peptide(Figure1A).Crystallographic screening resulted in the successful cocrystallization of Nurf55in complex with an H31–19peptide.After molecular replacement with the known structure of Nurf55(Song et al.,2008),the initial mF oÀDF c differ-ence map showed density for H3residues1–14in bothNurf55molecules in the crystallographic asymmetric unit. Figures1B–1E show the structure of H31–19bound to Drosophila Nurf55,refined to2.7A˚resolution(R/R free=20.1%and25.0%, Table1;Figure S1A,available online).The H3peptide binds to theflat surface of the Nurf55WD40propeller(Figure1B),subse-quently referred to as the canonical binding site(c-site)(Gaudet et al.,1996).The H3peptide is held in an acidic pocket(Figures 1C and1E)and traverses the central WD40cavity in a straight line across the propeller(Figure1B).Nurf55binds the H3peptide by contacting H3residues Ala1, Arg2,Lys4,Ala7,and Lys9.Each of these residues forms side-chain specific contacts with the Nurf55propeller(Figures 1D and1E).The bulk of the molecular recognition is directed toward H3Arg2and Lys4.Ala1sits in a buried pocket with its a-amino group hydrogen bonding to Nurf55Asp252,which recognizes andfixes the very N terminus of histone H3.The neighboring Arg2is buried deeper within the WD40propeller fold,with its guanidinium group sandwiched by Nurf55residues Phe325and Tyr185(Figure1D).H3Lys4binds to a well-defined surface pocket on Nurf55located on blade2,near the central cavity of the propeller.Its3-amino group is specifically coordi-nated by the carboxyl groups of Nurf55residues Glu183andGlu130and through the amide oxygen of Asn132(Figure1E).Lys9is stabilized by hydrophobic interactions on the WD40surface while having its3-amino group held in solvent-exposedfashion(Figure1D).Ser10of histone H3marks the beginningof a turn that inverses the peptide directionality.Histone H3residues Thr11–Lys14become progressively disordered andare no longer specifically recognized.No interpretable densitywas observed beyond Lys14.Taken together,Nurf55specificallyrecognizes an extended region of the extreme N terminus ofhistone H3(11residues long,700A˚2buried surface area)in thecanonical ligand binding location of WD40propeller domains. Structure of the Nurf55-Su(z)12Subcomplex of PRC2 The H3-Nurf55structure prompted us to investigate how Nurf55might bind histone tails in the presence of Su(z)12,its interactionpartner in PRC2(Nekrasov et al.,2005;Pasini et al.,2004).Asafirst step we mapped the Nurf55-Su(z)12interaction in detailby carrying out limited proteolysis experiments on reconstitutedDrosophila PRC2,followed by isolation of a Nurf55-Su(z)12subcomplex.Mass spectrometric analysis and pull-down exper-iments with recombinant protein identified Su(z)12residues73–143[hereafter referred to as Su(z)1273–143]as sufficient forNurf55binding(Figures S1C and S1D).Crystals were obtained when Drosophila Nurf55and Su(z)12residues64–359were set up in the presence of0.01%subtilisinprotease(Dong et al.,2007).After data collection,the structurewas refined to a maximal resolution of2.3A˚(Table1).Molecularreplacement with Nurf55as search model provided clear initialmF oÀDF c difference density for a13amino acid-long Su(z)12 fragment spanning Su(z)12residues79–91(Figures2A–2C).Thefinal model was refined to2.3A˚(R/R free=17.5%/20.9%)and verified by simulated annealing composite-omit maps(Fig-ure S1B).The portion of Su(z)12involved in Nurf55binding willhenceforth be referred to as the Nurf55binding epitope(NBE).The Su(z)12binding site on Nurf55is located on the side of thepropeller between the stem of the N-terminal a helix(a1)andthe PP loop(Figures2A and2B).Binding between Su(z)12andNurf55occurs mostly through hydrophobic interactions in anextended conformation.The interaction surface betweenNurf55and the NBE is large for a peptide,spanning around800A˚2.Sequence alignment between Su(z)12orthologs revealsthat the NBE is highly conserved(53%identity and84%similarity)in animals and in plants(Figure2E).With the exceptionof Su(z)12Arg85,the majority of the conserved Su(z)12NBEresidues engage in hydrophobic packing with Nurf55(Figures2B and2C).Together with the Su(z)12VEFS domain and theC2H2zincfinger(C5domain)(Birve et al.,2001),the NBE consti-tutes the only identifiable motif in Su(z)12found conserved in allSu(z)12orthologs.The NBE binding site on Nurf55has previously been shown tobe occupied by helix1of histone H4(Figure2D)(Murzina et al.,2008;Song et al.,2008),an epitope not accessible in assemblednucleosomes(Luger et al.,1997).Nurf55binds H4and theSu(z)12NBE epitope in a different mode,and importantly,withopposite directionality(Figure2D).The detailed comparison ofthe Nurf55-Su(z)12structure with that of H4bound to Nurf55Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3Molecular Cell42,330–341,May6,2011ª2011Elsevier Inc.331strongly suggests that binding of Su(z)12(NBE)and of H4(helix 1)are mutually exclusive (Figure 2D).We therefore refer to the Su(z)12and H4binding site on Nurf55as the S/H -site.Su(z)12fragments that include the NBE have poor solubility by themselves and generally require Nurf55coexpression for solu-bilization.However,we were able to measure binding of a chem-ically synthesized Su(z)1275–93peptide to Nurf55by isothermal titration calorimetry (ITC)and found that the peptide was bound with a K D value of 6.7±0.3m M in a 1:1stoichiometry (Fig-ure 2F).Pull-down experiments with recombinant proteinandFigure 1.Crystal Structure of Nurf55in Complex with a Histone H31–19Peptide(A)Nurf55binds to an H31–15peptide with an affinity of $0.8±0.1m M as measured by FP.It has similar affinity for an H31–31peptide (2.2±0.2m M)but no binding can be detected to an H319–38peptide.(B)Ribbon representation of Nurf55-H31–19.Nurf55is shown in rainbow colors and H31–19is depicted in green.The peptide is bound to the c -site of the WD40propeller.(C)Electrostatic surface potential representation (À10to 10kT/e)of the c -site with the H3peptide shown as a stick model in green.(D)Close-up of the c -site detailing the interactions between Nurf55(yellow)and the H31–19peptide (green),with a water molecule shown as a red sphere.(E)Schematic representation of interactions between the H31–19peptide (green)and Nurf55(yellow).Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3332Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.streptavidin beads suggest that Su(z)12residues 94–143harbor an additional Nurf55binding site not visible in the structure (Figure S1E).Su(z)12144–359,lacking the N-terminal 143residues,no longer binds to Nurf55.The NBE (residues 79–93)and the region adjacent to the NBE (residues 94–143)are thus required for stable interaction with Nurf55.The extended NBE was found enriched after limited proteolysis and in subsequent gel filtration runs coupled with quantitative mass spectrometry (Figure S1C).As the NBE was the only fragment visible after structure determi-nation,we conclude that it represents the major Su(z)12interac-tion epitope for Nurf55binding.The Nurf55-Su(z)12Complex Binds to Histone H3In order to study the potential interdependence of the identified Nurf55binding sites we compared binding of Nurf55and Nurf55-Su(z)12to the histone H3N terminus.FP experiments showed similar affinities for binding of a histone H31–15peptide to Nurf55(K D $0.8±0.1m M;Figure 1A)and a Nurf55-Su(z)1273–143complex (K D $0.6±0.1m M;Figure 2G).Importantly,mutation of Nurf55resi-dues contacting H3via its c -site drastically reduced binding to an H31–15peptide (Figure S2A),demonstrating that the Nurf55-Su(z)1273–143complex indeed binds the H31–15peptide through the c -site.We conclude that the presence of Su(z)12is compatible with Nurf55binding to H3via its c -site and that the two binding interactions are not interdependent.The observation that the Su(z)12NBE occupies the same Nurf55pocket that was previously shown to bind to helix 1of histone H4prompted us to test whether the Su(z)1273–143-Nurf55complex could still bind to histone H4.We performed pull-down experiments with a glutathione S-transferase (GST)fusion protein containing histone H41–48(Murzina et al.,2008)and found that H4stably interacted with isolated Nurf55but not with Su(z)1273–143-Nurf55(Figure 2H).In PRC2,the presence of Su(z)12in the Nurf55S/H -site therefore precludes binding to helix 1of histone H4.H3Binding by Nurf55-Su(z)12Is Sensitive to the Methylation Status of Lysine 4We next investigated how posttranslational modifications of the H3tail affect binding to the Nurf55-Su(z)1273–143complex.Modi-fications on H3Arg2,Lys9,and Lys14did not change affinity of Nurf55-Su(z)12for the modified H31–15peptide (Figures S2B and S2D).In contrast,peptides that were mono-,di-,or trimethylated on Lys4were bound with significantly reduced affinity exhibiting K D values of 17±3m M (H3K4me1),24±3m M (H3K4me2),and >70m M (H3K4me3),respectively (Figure 2I).The FP binding data were independently confirmed by ITC measurements (Figures S2C–S2F).Together,these findings are in accord with the structural data,which show that H3K9and H3K14are being held with their 3-amino moiety solvent-exposed,while the H3K4side chain is tightly coordinated (Figure 1E).The additional methyl groups on the H3K43-amino group are expected to progressively decrease affinity because of increased steric clashes within the H3K4binding pocket.H3K27Methylation by PRC2Is Inhibited by Histone H3K4me3MarksWe then examined the effect of H3K4me3modifications,which are no longer retained by Nurf55-Su(z)12,on the catalytic activity of PRC2.In a first set of experiments,we determined PRC2steady-state parameters on histone H31–45peptide substrates that were either unmodified or methylated at Lys 4.We observed similar K M values for H3and H3K4me3peptides of 0.84±0.21m M and 0.36±0.07m M,respectively (Figure 3A),and similar K M values for SAM (5.42±0.65m M for H3and 10.04±1.56m M for H3K4me3).The turnover rate constant k cat ,however,was 8-fold reduced in the presence of H3K4me3:2.53±0.21min -1for unmodified H3and 0.32±0.08min -1in the presence of H3K4me3(Figure 3A).While substrate binding is largely unaf-fected,turnover is thus severely inhibited in the presence of H3K4me3.This behavior,which results in a k cat /K M specificity constant of 7.83103M -1s -1(unmodified H3)compared to 0.533103M -1s -1(H3K4me3),is consistent with heterotrophic allosteric inhibition of the PRC2HMTase triggered by the pres-ence of the H3K4me3.To investigate the effect of the H3K4me3modification on PRC2activity in the context of nucleosomes,we reconstituted mononucleosomes with a trimethyllysine analog (MLA)at Lys4in H3(referred to as H3Kc4me3;Figure S3A)(Simon et al.,2007).We found that total H3K27methylation (measured by incorporation of 14C-labeled methyl groups)was substantially impaired on H3Kc4me3-containing nucleosomes compared to wild-type nucleosomes (Figures S3B and S3C).We used western blot analysis to monitor how levels of H3K27mono-,di-,and trimethylation were affected by the H3Kc4me3modifica-tion.While H3K27me1formation was reduced by more than 50%on H3Kc4me3nucleosomes compared to unmodified nucleosomes (Figures 3B and 3C),H3K27dimethylationandTable 1.Crystallographic Data and Refinement StatisticsNurf55–Su(z)12Nurf55–H31–19Space Group P212121P212121theses.Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.333titrant H3:S/H-siteN CNCSu(z)12 79-91Histone H4 31-41PP-loophelix α10.00.51.0 1.52.0 2.5-16.00-14.00-12.00-10.00-8.00-6.00-4.00-2.000.00-2.00-1.50-1.00-0.500.00020406080100120Time (min)µc a l /s e cMolar RatioK C a l /M o l e o f I n j e c t a n tK D = 6.7 ± 0.3 µMtitrant: H31-15DEFGH ICA B protein concentration [µM]Nurf55-Su(z)12protein concentration [µM]00.20.40.60.81.000.20.40.60.81.00.010.11101000.010.1110100Nurf55Nurf55-Su(z)12unmod K4me1K4me2K4me3f r a c t i o n b o u n df r a c t i o n b o u n d G ST -H 41-48 + N u r f 55G S T -H 41-48 + N u r f 55-S u (z )1273-143N u r f 55(m o c k c o n t r o l )N u r f 55-S u (z )1273-143(m o c k c o n t r o l )Nurf55GST-H41-48Su(z)1273-143i np ut i n p u t i n p u t i n p u t G S T -p u l l d o w n G S T -p u l l d o w n G S T -p u l l d o w n G S T -p u l l d o w n Figure 2.Crystal Structure and Characterization of Nurf55in Complex with the Su(z)12Binding Epitope for Nurf55(A)Ribbon representation of Nurf55-Su(z)12.Nurf55(rainbow colors)depicts the WD40domain nomenclature and Su(z)12is shown in magenta.The S/H -site is marked by a dashed box.(B)Detailed interactions of Su(z)12(magenta)with the S/H -site (yellow).Water molecules are depicted as red spheres.(C)Schematic representation of interactions between Su(z)12(magenta)and Nurf55(yellow).(D)Overlay of the backbone trace of Su(z)12(magenta)and the H4helix a 1(orange)(Song et al.,2008)in the S/H -site.(E)Alignment of the Su(z)12NBE with sequences from Drosophila melanogaster (dm,Q9NJG9),mouse (mm,NP_954666),human (hs,AAH15704),Xenopus tropicalis (xt,BC121323),zebrafish (dr,BC078293),and the three Arabidopsis thaliana (at)homologs Fis2(ABB84250),EMF2(NP_199936),and VRN2(NP_567517).Identical residues are highlighted in yellow.(F)ITC profile for binding of a Su(z)1275–93peptide to Nurf55.Data were fitted to a one-site model with stoichiometry of 1:1.The derived K D value is 6.7±0.3m M.(G)Binding of H31–15to Nurf55(0.8±0.1m M)and Nurf55-Su(z)1273–143(0.6±0.1m M)measured by FP.Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3334Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.trimethylation were impaired by more than 80%by using H3Kc4me3nucleosomes (Figure 3C).In order to ascertain that inhibition of PRC2is indeed due to trimethylation of the aminogroup in the lysine side chain,and not due to the use of the MLA,we performed HMTase assays on H3K4me3-containing nucleosomes generated by native peptide ligation (Shogren-Knaak et al.,2003)and on H3Kc4me0and H3K4A nucleosomes.H3K27mono-,di-,and trimethylation was comparably inhibited on H3K4me3and on H3Kc4me3-containing nucleosomes,but was not affected by H3Kc4me0and H3K4A (Figures S3D and S3E).We conclude that H3K4me3specifically inhibits PRC2-mediated H3K27methylation with the most pronounced inhibi-tory effects observed for H3K27di-and trimethylation.We next tested whether the H3K4me3modification affects PRC2nucleosome binding.In electrophoretic mobility shift assays (EMSA),we found that PRC2binds unmodified or H3Kc4me3-modified nucleosomes with comparable affinity (Fig-ure S4A).Even though binding of Nurf55to the N terminus of ABC571141712290286571141712290286unmodified H3Kc4me3H3K27me3H4dmPRC2 [nM]H3K27me2H4H3K27me1H4H3K27me1H3K27me3H3K27me2dmPRC2 + unmod H3dmPRC2 + H3Kc4me3r e l a t i v e H M T a s e a c t i v i t ySubstrate Apparent Km of SAM (µM)Apparent Km of peptide (nM)k cat (min )-1k cat /Km (M S )-1-1H31-45 -biotin H3K4me31-45 -biotin5.42 ± 0.6510.04 ± 1.56355 ± 74.60.32 ± 0.080.53 x 10836 ± 207 2.53 ± 0.217.8 x 1033Figure 3.HMTase Activity of PRC2Is In-hibited by H3K4me3Marks(A)HMTase assay with PRC2and H31–45-biotin peptides measuring the concentration of SAH produced by the enzymatic reaction.When an H3K4me3-modified peptide is used,the specificity constant (k cat /K M )is drastically reduced,indicative of heterotrophic allosteric inhibition.(B)Western blot-based HMTase assay by using recombinant Drosophila mononucleosomes (571nM)and increasing amounts of PRC2.HMTase activity was monitored with antibodies against H3K27me1,H3K27me2,or H3K27me3as indicated;in each case the membrane was also probed with an antibody against unmodified histone H4to control for equal loading and western blot processing.Deposition of K27di-and trime-thylation is drastically reduced when nucleosomes are used that carry a H3Kc4me3modification.(C)Quantification of HMTase activity of Drosophila PRC2(286nM)on unmodified and H3Kc4me3-modified nucleosomes by quantitative western blotting.histone H3is almost 100-fold reduced byH3K4me3(Figure 2I and Figure S2F),inter-action of the Nurf55c -site with H3K4does not seem to make a detectable con-tribution to nucleosome binding by PRC2in this assay.Consistent with the allosteric mechanism of H3K4me3inhibition that we had observed in the peptide assays (Figure 3A),inhibition of the PRC2HMTase activity by H3K4me3-containingnucleosomes is not caused by impaired nucleosome binding,but is rather the consequence of reduced catalytic turnover.H3K4me3Needs to Be Present on the Same Tail as K27to Inhibit PRC2We then assessed whether inhibition of the PRC2HMTase activity by H3K4me3requires the K4me3mark to be located on the substrate nucleosome (in cis ),or whether it could also be trig-gered if the H3K4me3modification was provided on a separate peptide (in trans ).We performed HMTase assays on unmodified oligonucleosomes in the presence of increasing amounts of a histone H31–15peptide trimethylated at K4(H31–15-K4me3)(Figure 4A).Addition of the H31–15-K4me3peptide did not affect PRC2HMTase activity at peptide concentrations as high as $200m M.When testing H31–19-unmodified peptide in controls at comparable concentrations,we did observe concentration-dependent PRC2inhibition (Figure 4A),probably because of substrate competition at large peptide excess.As H3K4me3-(H)GST pull-down assay with recombinant GST-H41–48and Nurf55and Nurf55-Su(z)1273–143proteins.GST-H41–48is able to bind Nurf55alone but in the Nurf55-Su(z)1273–143complex the binding site is occupied by Su(z)12(left panel).Control pull-downs with GST beads and either Nurf55or Nurf55-Su(z)1273–143alone showed no unspecific binding (right panel).(I)Binding of different H31–15peptides to Nurf55-Su(z)1273–143measured by FP.While unmodified H3is bound with 0.8±0.1m M affinity,methylation of Lys 4drastically reduces binding affinity (17±3m M for K4me1,24±3m M for K4me2,and >70m M for K4me3).Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.335modified peptides did not show this competitive behavior,we conclude that PRC2is not inhibited by H3K4me3in trans and that H3K4me3and unmodified H3peptides are probably bound to PRC2in a different fashion.Analogously,we saw no inhibition when testing the effect of H3K4me3in trans by using peptides as substrates (Figure S4B ).Taken together,our findings strongly argue that H3K4me3only inhibits PRC2if present on the same tail that contains the H3K27target lysine (in cis ).Previous studies reported that addition of H3K27me3peptides in trans enhances H3K27methylation of oligonucleosomes by human PRC2through binding to the EED WD40domain (Margueron et al.,2009;Xu et al.,2010).We tested whether addition of H3K27me3peptides in trans would stimulate H3K27methylation by PRC2on H3Kc4me3-modified nucleosomes.We observed that the inhibitory effect of H3Kc4me3-containing nucleosomes can,at least in part,be overcome through addition of high concentrations of H3K27me3peptides (Figure 4B and Figure S4C).PRC2is therefore able to simultaneously integrate inhibitory (H3K4me3)and activating (H3K27me3)chromatin signatures and adjust its enzymatic activity in response to the surrounding epigenetic environment.PRC2Inhibition of H3K4me3Is Conserved in Mammalian PRC2Our results with Drosophila PRC2prompted us to investigate to what extent inhibition by H3K4me3is an evolutionarily conserved mechanism.H3K27methylation by human and mouse PRC2on nucleosome substrates carrying H3Kc4me3modifications was also strongly inhibited,comparable to the inhibition observed for Drosophila PRC2(Figure 5A and Figure S5A).The Su(z)12Subunit Codetermines Whether PRC2Is Inhibited by H3K4me3In Arabidopsis thaliana ,three different E(z)homologs combined with three Su(z)12homologs have been described.The distinct PRC2complexes in plants harboring the different E(z)or Su(z)12subunits are implicated in the control of distinct developmental processes during Arabidopsis development (He,2009).In this study we focused on PRC2complexes con-taining the E(z)homolog CURLY LEAF (CLF).We expressed and reconstituted the Arabidopsis PRC2complex comprising CLF,FERTILIZATION INDEPENDENT ENDOSPERM (FIE,a homolog of ESC),EMBRYONIC FLOWER 2(EMF2,a homolog of Su(z)12),and MULTICOPY SUPPRESSOR OF IRA (MSI1,a homolog of Nurf55).We found that CLF indeed functions as a H3K27me3HMTase (Figure 5B).Moreover,H3K27methylation by the CLF-FIE-EMF2-MSI1complex on nucleosome arrays containing H3Kc4me3was inhibited (Figure 5B)in a manner comparable to human or Drosophila PRC2.We next tested a related Arabidopsis PRC2complex again composed of CLF,FIE,and MSI1but containing the Su(z)12homolog vernalization 2(VRN2)instead of EMF2.The VRN2protein is specifically implicated as a repressor of the FLC locus,thereby controlling flowering time in response to vernalization (reviewed in Henderson and Dean,2004).The CLF-FIE-MSI1-VRN2complex was active on unmodified nucleosomes but,strikingly,it was not inhibited on H3Kc4me3-modified nucle-osomes (Figure 5C).Substitution of a single subunit (i.e.,EMF2by VRN2)thus renders the complex nonresponsive to the H3K4methylation state.While PRC2inhibition by H3K4me3appears hardwired in mammals and flies,in which only a single-Su(z)12ortholog is present,Arabidopsis inhibition can be enabled or disabled through exchange of the Su(z)12homolog.The Su(z)12C Terminus Harboring the VEFS Domain Is the Minimal Su(z)12Domain Required for Activation and Active Mark InhibitionThe importance of the Su(z)12subunit in active mark H3K4me3inhibition prompted us to map the Su(z)12domains required for inhibition.Previous findings showed that E(z)or E(z)-ESC in the absence of Su(z)12is enzymatically inactive (Nekrasov et al.,2005).Moreover,the VEFS domain (Birve et al.,2001)was found to be the major E(z)binding domain (Ketel et al.,2005).We reconstituted mouse PRC2complexes containing EZH2,EED,and either SUZ12C 2H 2domain +VEFS (residues 439–741)or SUZ12VEFS alone (residues 552–741).Both of these minimal complexes were active in HMTase assays on nucleosomes (Figure 5D)but with lower activity than that of the full PRC2complex.We therefore focused on formation ofAB26511030206H3K27me3H4peptides in trans[µM]H3K27me2H4H3K27me1H4H3 unmod H3K4me3H3K36me326511032062651103206n o e n z ym e26511030H3K27me3H4peptides in trans[µM]H3K27me2H4H3K27me1H4H3K27me3n oe n z y m eFigure 4.PRC2Activity Is Not Inhibited by H3K4me3Peptides in trans(A)Western blot-based HMTase assay by using unmodified 4-mer oligonu-cleosomes (36nM)and increasing amounts of H3peptides added in trans .Enzyme concentration was kept constant at 86nM.Western blots were pro-cessed as described in Figure 3B.HMTase activity is inhibited by an unmodified H31–19peptide (left),but not by H3K4me3-or H3K36me3-modified peptides.(B)HMTase assay with H3Kc4me3-modified oligonucleosomes (36nM),86nM PRC2,and H3K27me3peptide in trans .Western blots were processed as described in Figure 3B.HMTase activity of PRC2can be stimulated by the H3K27me3peptide even on inhibiting substrate leading to increased levels of H3K27di-and trimethylation.Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3336Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.。
槲皮素-罗丹明6G.荧光猝灭体系研究

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w t l=2 6 × 1 ・mo ~ , 1= 1 4 a d k i k h . 3 0L l n .1 n 2= 7 4 × 1 ・mo ~ ,n . 2 0L l 2= 1 0 . 7. T o l e r w i a n r lt n h p e e g tb t e 0 F a d t e c n e t t n o u r e i n t e r n e f . 1~0 21 。 e ai s i s w r o ewe n F / n h o c nr i fq e c t i a g so 0 o ao n h 0 . x g mL。 a d 0 2~5 0 x ・mL~ r s e t ey w t h ee t n l to . n ・ 。 n . . 1 g e p c i l i t e d tc i i f 9 g mL一 n e r c v r f v h o mi 5 a d t e o e y o h 9 . % ~1 4 . T e me o al e u e o u n i t e d t r n t n o u r ei . 17 0 % h t d C l b s d fr q a t ai ee mi ai fq ec t h t v o n Ke r s q e c t y wo d : u r ei n;r o a n G ;f o e c n e q e c i g h d mi e6 l r s e c u n h n ;me h n s u cai m
氯化铁氧化掺杂的聚苯胺纳米纤维团簇

* 2006-04-24 收稿, 2006-05-26 修稿; 国家自然科学基金( 基金号 50533030) 资助项目; ** 通讯联系人, E-mail: wanmx@ iccas. ac. cn 462
5期
丁杭军等: 氯化铁氧化掺杂的聚苯胺纳米纤维团簇
463
Generator 作恒 流电 源流, KEITHLEY 196 SYSTEM DMM 测其电压. 112 聚苯胺纳米纤维的制备
高, 极化子的吸收峰出现蓝移, 说明其产物的氧化 程度增强. 这说明在不同 [ FeCl3] P[ An] 比例的条 件下所得的 PANI 纤维均 为掺杂态. 与红外吸收 光谱的分析结果一致.
在界面体系中, 苯胺分散于下层有机相中, 氧 化剂溶于上层水溶液中, 当两种溶液混合后, 在两
Fig. 1 Typical SEM images of PANI-FeCl3 nanofibers prepared at different [ FeCl3 ]P[ An] ratios a) 1B1; b) 2B1, c) 6B1; d) 8B1
采用扫描电镜( SEM, Hitach-i 530) , 透射电镜 ( TEM, Hitachi, H- 9000 ) , 傅 立 叶 红 外 ( FT IR, Eouninox55, Bruker ) , 紫外可见吸 收( UV, V- 570 型 紫外 吸 收 分 光 广 度 计 ) , X- 射 线 衍 射 ( XRD, DPmax- 2400) , X- 射 线 光 电 子 能 谱 ( XPS, ES- 300, Kratos) 等手段对产物进行结构分析和表征. 电导 率是 采 用 四 电 极 法 ( four- probe method) 测 定, ADVATES TR6142 Programmable DC VoltagePCurrent
新型聚醚聚氨酯微孔弹性体的研制

科研开发
弹性体, , ( ) : ! " " # $ " % $ ! & ’ # ( ’ ( ! ’ ) * + , . / 0 . 1 2 34 / 5 , * 1
新型聚醚聚氨酯微孔弹性体的研制 !
亢茂青, 殷 宁, 冯月兰, 赵雨花, 瞿 波, 王心葵
(中国科学院 山西煤炭化学研究所, 山西 太原 " ) # " " " ’
第 !期
亢茂青, 等1 新型聚醚聚氨酯微孔弹性体的研制
・> .・
善了相分离程度, 玻璃化转变温度低, 这将为制备 高质量聚醚聚氨酯制品提供了必要的原料来源。
! 实验部分
! " ! 原材料 , 二苯基甲烷二异氰酸酯 ($% ) : 烟台万 ! ! " # & 、 华产品; 普通聚醚多元醇 (传统碱催化) : ’ % # ( ) 天 津 第 三 石 油 化 工 厂 产 品; 新型高活性 * * + ,, (伯羟 基 质 量 分 数 为 ) ) 、 低 不饱和度 + - !) . 0 (/ ! / 聚 醚 多 元 醇: 、% + 1 + + .2 2 3 4 6 7 7 # ( ( 5) [8 (( / , , ] , 9 :0 (;() 2 < 8 9 $ = " .> + + ? 0( 5 5 、 [8 (( / , @ % 7 7 # ( ) 9 :0 );() 2 < 8 9 $ =" 5 5 , ] , 、 [ ( ) / , ! + + A? 0 ( BC # ( ( 8 9 :0 ( ( ; (2 < 8 9 5 5 , , 、 [8 (( $ = "AD . + ?0*] E C # ( ) 9 :0 );() / ,$ , , 、C$, 2 < 8 9 ="D+ > + ?0*] F # * + + + 5 5 [8 (* ) / ,$ , , 9 :0 .;( 2 < 8 9 ="*( + D ? 0( 5 5 质量分数为 ] 均为自制; 接枝聚合物聚醚 ’ + > . , 天津第三石油化工厂产品; 扩 多元醇 : G 8 G H * ( ) 链剂 > , 丁二醇 (I : 北京化学试剂厂产品; ! # % 8) : 质量分数为* 催化剂 % 6 @ B 3* * 7 J * -三亚乙基二 胺 K 质量分数为 D 的一缩二乙二醇, 美国气 A 体产品公司产品; 有机硅匀泡剂: 、 % 6 @ B 3% /* + ! * 、 , 美国气体产品公司 % 6 @ B 3% / * + ! ( % 6 @ B 3% / > H * (溶剂型) : 青岛德慧精细 产品; 脱模剂 % 9 # * A ( ( 化工有限公司产品; 改性剂: 自配; 稳定剂: 有机 酸, 北京化学试剂厂产品。 ! " # 制备 ! " # " ! 鞋底原液制备 采用半预聚体法合成聚醚聚氨酯微孔弹性体 的原料分为 L、 扩链 I 组份。L 组份系多元醇、 剂、 发泡剂、 催化剂、 表面活性剂等组份混合而成 的组合料。I 组份为异氰酸酯封端的半预聚体。 真空脱 聚醚多元醇在 > ( +M 左右的条件下, 水>N , 密封保存以备用。 (多元醇组份) > 1 ( 1 > 1 > L 组份 将脱水聚醚多元醇、 扩链剂、 表面活性剂、 催 化剂等按一定比例加入三口烧瓶中, 升温至 D + ! 充分混合> , 混合均匀后, 再冷却至* A +M, ! (N . 出料, 密封保存。 ! ! +M (异氰酸酯半预聚体) > 1 ( 1 > 1 ( I 组份 将计量的异氰酸酯与脱水聚醚多元醇及改性 剂在氮气保护下加入三口烧瓶中, 在) +M左右反 应( , 真空脱除气泡, 自然降温 (退火处理) 、 ! *N 出料, 分析游离的—, 密封保存。 / 8 质量分数,
通过醚的α-位C(sp^(3))—H键官能化合成醚类衍生物的研究进展

第6期
" 马金凤等:通过 的!位C#8 )—H键官能化合成"类衍生物的研究进展
一 539 —
Scheme 2
仅
的有机化合物中,还
布
物、药物和其他生物活性 等复杂
中。例如 裸甲藻产生的神经毒素(Breve toxin B)、治疗丙型肝炎病毒(HCV)感 的Sofos-
buvir、兴奋剂Zylofuramino-"肾上腺素 拮抗剂 Idazoxan 等(ChaN 1) [3_6] o 因此,通过"的!烯
R1 rt, dark, 1 h
w。丫為」
R1
Scheme 13
同年,该课题组[12]又采用耙催化,实现了酰
腺、芳基卤化物和环"的三组分交叉偶联
# Schema 6a)。该反应由于产物1a和2a的极性
接近,无法
色
, 其又将
混合产物进一步氢化,获
净的产物
#2a,Schema 6a)。 2020年,刘 涛课题组[13]发表了钻催化的
化和
程一步形成C—CSC=O键,从而
获得中等 好的收率酮产物。反应的机理研究
明,
K2S2O8夺 "!C(83)—H
的上H,反应历 基 而完成。
2017 王伟课题组[17]报
的无金
化的交叉
联(CDC)反应, 应
7-位基琥珀
(NHS) 介导物,直接将多
种"连到杂 上(Scheme 11 )。NHS介导的
—541 —
Sheeme6
1 mol% Co(acac)2,5 equiv TBHP 2 equiv DABCO , 70 °C, 12 h
Sheeme7
最新SCI期刊目录(6606种SCI期刊,期刊名缩写、全名、ISSN、学科分区大类加小类、

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CHROMATOGRAPHY0065-24150108-7681 ACTA CRYSTALLOGR ACTA CRYSTALLOGRAPHICA SECTION B-STRUCTURAL SCIEAUST J CHEM AUSTRALIAN JOURNAL OF CHEMISTRY0004-94251386-2073 COMB CHEM HIGH T COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENJ FLUORESC JOURNAL OF FLUORESCENCE1053-0509 POLYM DEGRAD STA POLYMER DEGRADATION AND STABILITY0141-3910 EUR POLYM J EUROPEAN POLYMER JOURNAL0014-3057 PURE APPL CHEM PURE AND APPLIED CHEMISTRY0033-45451010-6030 J PHOTOCH PHOTOB JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A-CHEJ ORGANOMET CHEM JOURNAL OF ORGANOMETALLIC CHEMISTRY0022-328X MACROMOL CHEM PH MACROMOLECULAR CHEMISTRY AND PHYSICS1022-1352 CARBOHYD POLYM CARBOHYDRATE POLYMERS0144-8617 J SOLID STATE CH JOURNAL OF SOLID STATE CHEMISTRY0022-4596 ADV HETEROCYCL C ADVANCES IN HETEROCYCLIC CHEMISTRY0065-2725 ACTA CRYSTALLOGR ACTA CRYSTALLOGRAPHICA SECTION A0108-7673 MICROCHEM J MICROCHEMICAL JOURNAL0026-265X CHEM PHYS CHEMICAL PHYSICS0301-0104 ORG PROCESS RES ORGANIC PROCESS RESEARCH & DEVELOPMENT1083-6160 CATAL LETT CATALYSIS LETTERS1011-372X J CRYST GROWTH JOURNAL OF CRYSTAL GROWTH0022-0248 INORG CHEM COMMU INORGANIC CHEMISTRY COMMUNICATIONS1387-7003 VIB SPECTROSC VIBRATIONAL SPECTROSCOPY0924-2031 SURF SCI SURFACE SCIENCE0039-6028 J ANAL APPL PYRO JOURNAL OF ANALYTICAL AND APPLIED PYROLYSIS0165-2370 USP KHIM+USPEKHI KHIMII0042-1308 POLYHEDRON POLYHEDRON0277-5387 CARBOHYD RES CARBOHYDRATE RESEARCH0008-6215 SUPRAMOL CHEM SUPRAMOLECULAR CHEMISTRY1061-0278 INORG CHIM ACTA INORGANICA CHIMICA ACTA0020-1693 MINI-REV ORG CHE MINI-REVIEWS IN ORGANIC CHEMISTRY1570-193X SOLID STATE SCI SOLID STATE SCIENCES1293-2558 COLLOID SURFACE COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGI0927-7757 MICROCHIM ACTA MICROCHIMICA ACTA0026-3672 ADV QUANTUM CHEM ADVANCES IN QUANTUM CHEMISTRY0065-3276 J MOL MODEL JOURNAL OF MOLECULAR MODELING1610-2940 POLYM INT POLYMER INTERNATIONAL0959-8103 CURR ANAL CHEM Current Analytical Chemistry1573-4110 ANAL SCI ANALYTICAL SCIENCES0910-6340 CHEM LETT CHEMISTRY LETTERS0366-7022 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6.0365.36 5.7317.325.712 5.287 5.6464.189 4.086 4.1154.097 4.009 3.9023.952 3.959 3.796.802 5.93 4.2845.276.394 4.7895.485 5.827 5.0685.454 5.33 5.0155.619 4.977 4.7625.34 5.141 4.5213.5714.176 6.855.128 4.802 4.6595.493 4.354 4.634.6045 4.7314.542 4.836 4.1926.511 4.041 2.8934.214 4.6 3.7924.215 4.046 4.3394.197003.394.297 4.8934.274 4.308 3.6275.333 3.074 3.794.147 4.123 3.9114.146 4.169 3.6643.815 3.833 3.6323.756 3.641 3.5544.028 3.269 3.633.821 3.529 3.4053.535 3.468 3.7293.636 3.502 3.4493.761 3.553 3.1983.184 3.961 3.2324.064 3.343 2.892 3.6 3.325 3.303 3.39600 3.181 3.664 3.307 3.643 2.986 3.423 3.58 3.212 3.012 3.212 3.629 2.495 3.477 2.641 3.583 3.55 3.167 2.874 3.206 3.374 2.81 3.011 3.154 3.153 3.146 3.186 2.894 3.331 3.065 2.773 3.19 3.166 2.63 2.94 3.062 2.945 2.424 3.333 3.0832.871 2.9183.0473.328 2.867 2.5912.9773.085 2.6633.016 2.914 2.769 2.897 2.869 2.817 2.61 2.8443 2.772 2.971 2.68 2.901 2.949 2.444 2.659 2.763 2.8382.942 2.651 2.6473.5 2.973 1.6562 2.11843.5 2.582 2.814 2.707 2.5112.694 2.597 2.7043.004 2.764 2.148 2.746 2.632 2.535 2.796 2.634 2.468 2.538 2.615 2.509 2.555 2.21 2.7961.8332.66732.48400 2.882 2.301 2.187 2.37 2.537 2.446 2.938 1.754 2.5 2.796 2.434 1.96 2.791 2.394 1.878 2.47 2.257 2.333 2.443 2.309 2.233 2.212 2.36 2.3212.169 2.207 2.462 2.6 1.615 2.6 2.341 2.163 2.172 2.405 2.36 1.895 1.747 2.344 2.551.882.101 2.612.32 2.073 2.174 2.143 2.248 2.113 2.237 2.232 1.92 2.362 1.911 2.0981.8662.168 2.3322.202 2.046 2.021 2.644 1.782 1.784 1.91 2.149 2.1071.352.15 2.6472.051 2.385 1.676 2.505 1.8 1.558 1.961 1.805 1.984 1.905 1.833 2.004 1.867 1.883 1.772 1.757 1.95 1.809 1.854 1.85 1.787 1.81 1.78 1.88 1.731 1.855 1.88 1.911 2.12 1.412 1.832 1.893 1.717 1.801 1.756 1.843 1.96 1.723 1.703 1.846 1.64 1.861 1.94 1.713 1.674 1.4492 1.768 1.742 1.698 1.752 1.926 1.601 1.611 1.91 1.959 1.2373 1.2750.83 2.018 1.669 1.384 2.029 1.557 1.475 1.633 1.815 1.5 1.735 1.508 1.589 1.478 1.48 1.734 1.529 1.533 1.6 1.659 1.562 1.417 1.593 1.513 1.515 1.736 1.62 1.249 1.415 1.594 1.593 1.677 1.404 1.505 1.594 1.486 1.4951.63 1.483 1.438 1.443 1.434 1.61 1.396 1.515 1.55 1.53 1.352 1.4321.443 1.44 1.4192.215 1.3890.644 1.415 1.367 1.342 1.456 1.421 1.146 1.529 1.305 1.145 1.43 1.539 1.003 1.132 1.362 1.417 1.317 1.368 1.182 1.4330.888 1.51 1.272 1.036 1.427 1.27 1.224 1.233 1.295 1.229 1.162 1.281 1.3620.986 1.312 1.145 1.171 1.102 1.26 1.241 1.205 1.204 1.153 1.311 1.245 1.325 1.133 1.084 1.126 1.153 1.251 1.187 1.008 1.306 1.37 1.220.87 1.435 1.0570.965 1.377 1.2530.8 1.241 1.124 1.026 1.257 1.1560.95 1.084 1.21 1.068 1.189 1.023 1.115 1.4260.9720.92 1.167 1.112 1.016 1.1880.982 1.106 1.140.835 1.1760.98 1.066 1.0771.127 1.0220.969 1.2520.940.841 1.2650.9780.767 1.1550.9840.838 0.9810.977 1.001 0.9150.981 1.0040.745 1.032 1.121.1350.8690.880.9460.886 1.0141.0260.9770.825 1.2830.9190.6260.9820.683 1.153 0.9970.8380.918 0.909 1.0930.7030.8820.9340.8681.1140.7980.76 0.7320.8670.978 0.6330.923 1.009 0.9210.7680.863 0.7840.8790.881 0.8910.910.720.6610.9170.9341.0320.8420.616 0.8990.8130.776 0.650.9170.914 0.6210.746 1.109 0.8520.770.825 0.9780.750.679 0.9450.7190.712 0.6820.8440.783 0.720.7590.8 0.7820.6830.798 0.860.6790.699 0.6210.8540.743 0.6180.8570.742 0.8310.6060.778 0.5610.7190.896 0.6620.7660.738 0.7410.6550.767 0.5170.7320.89 0.6430.6960.729 0.4950.7120.85 0.7410.7540.561 0.6090.730.7120.5910.6280.8321.0720.5730.386 0.7270.6540.646 0.4670.372 1.174 0.5920.6950.724 0.770.6430.577 0.80.7670.353 0.6440.7530.506 0.560.7290.611 0.5750.6850.631 0.6920.6690.52 0.6810.7110.482 0.7670.6540.429 0.6730.6110.5610.5740.5970.658 0.5890.70.516 0.3850.6070.8 0.8240.3860.578 0.5520.6150.617 0.5140.6470.555 0.6620.6030.444 0.610.5840.514 0.5930.6830.431 0.5320.5690.583 0.6590.4990.509 0.5640.5290.56 0.5620.4960.54 0.5430.6960.333 0.5650.5410.466 0.6110.5360.423 0.5370.5540.478 0.7730.4850.305 0.5570.5110.492 0.5110.5280.52 0.5770.5520.418 0.5380.560.439 0.4690.5380.505 0.6330.5130.361 0.5180.4830.491 0.5340.5340.418 0.7580.3670.36 0.560.4220 0.380.460.609 0.5190.4570.473 0.3670.5080.567 0.1740.360.9 0.5790.4810.345 0.4240.3930.581 0.610.3770.378 0.5650.4730.322 0.3550.4340.547 0.4660.3680.491 0.470.4660.374 0.4680.4420.393 0.530.3980.368 0.440.3270.459 0.4810.380.358 0.40400 0.4750.4770.251 0.400 0.4170.590.1810.4080.3920.363 0.4880.3360.266 0.2310.4420.414 0.1160.3750.594 0.2790.40.397 0.440.3750.25 0.3840.330.34 0.2910.3060.443 0.3990.3040.307 0.3890.2610.287 0.2720.340.273 0.3020.2580.269 0.2950.2650.254 0.220.3310.225 0.2280.2860.262 0.2840.2620.208 0.2910.2690.187 0.2680.2920.173 0.1980.270.206 0.2440.1770.188 0.370.0970.133 0.170.1690.169 0.2210.0950.152 0.0970.1450.21 0.1520.1730.051 0.11000002006-2008年平均影响因子24.1343333315.1676666714.8153333314.7303333313.11811.7923333311.34510.380666679.3163333338.0027.9096666677.8906666677.067.0163333336.1375.5483333334.134.0026666673.9003333335.6725.4843333335.465.2663333335.1193333335.0006666674.8656666674.8634.8256666674.7783333334.5233333334.4816666674.2024.24.1974.1933333334.0696666674.0656666674.0603333333.9933.763.6503333333.6423333333.5853.5773333333.5293.5043.4593.433 3.4093333333.3963.384 3.3506666673.2683.112 3.2336666673.1973.133.106 3.075333333 3.056333333 2.995333333 2.982333333 2.946666667 2.945333333 2.928666667 2.908333333 2.8996666672.8612.818 2.807666667 2.764666667 2.753333333 2.746666667 2.7096666672.706 2.693333333 2.6773333332.665 2.638666667 2.637666667 2.6326666672.554 2.5203333332.52.484 2.4566666672.451 2.397333333 2.396666667 2.354333333 2.353333333 2.328333333 2.2976666672.279333333 2.271666667 2.2253333332.22 2.2136666672.1972.1892.168 2.129666667 2.1236666672.122 2.0896666672.07 2.0553333332.049 2.037333333 1.954333333 1.9166666671.914 1.840666667 1.838666667 1.830333333 1.8233333331.822 1.8143333331.8141.8 1.795333333 1.782333333 1.7756666671.739 1.730666667 1.7126666671.702 1.701666667 1.6903333331.687 1.649333333 1.6106666671.5641.5541.546 1.5403333331.5351.534 1.5286666671.525。
肾小管上皮细胞来源包含miroRNA-21的微囊泡在心肌肥大中的作用

㊃论著㊃基金项目:常州市卫计委重大项目肾脏来源包含m i r o R N A -21的微囊泡在心肌肥大中的作用及机制研究(Z D 201706)通信作者:赵家璧,E m a i l :z h a o ji a b i 1981@163.c o m 肾小管上皮细胞来源包含m i r o R N A -21的微囊泡在心肌肥大中的作用狄 佳1a ,周 华1a ,李 旻1a ,徐 婷1b,赵家璧2(1.苏州大学第三附属医院a .肾内科;b .免疫风湿科,江苏常州213003;2.南京医科大学附属常州第二人民医院病理科,江苏常州213003) 摘 要:目的 探讨肾小管上皮细胞来源包含m i r o R N A -21(m i R -21)的微囊泡(m i c r o v e s i c l e s ,MV s)在心肌肥大中的作用及机制㊂方法 用转化生长因子(t r a n s f o r m i n gg r o w t hf a c t o r ,T G F )-β1刺激肾小管上皮细胞,采用差速离心的方法提取条件培养液中的MV s ,D i l -C 18染料标记肾小管上皮细胞并用荧光显微镜观察受体心肌细胞,实时荧光定量聚合酶链式反应(q P C R )检测m i R -21含量,目镜测微尺测量心肌细胞长径,二喹啉甲酸(B C A )蛋白试剂盒及人心钠肽(A N P )试剂盒检测细胞蛋白含量及A N P 水平㊂预先用m i R -21i n h i b i t o r 转染受体心肌细胞,观察对细胞长径㊁蛋白含量及A N P 水平的影响㊂结果 T G F -β1可诱导供体肾小管上皮细胞产生MV s 并进入受体心肌细胞,使心肌细胞长径㊁蛋白含量㊁A N P 水平明显增加㊂T G F -β1诱导供体肾小管上皮细胞产生的MV s 中含有m i R -21,并可诱导受体心肌细胞肥大,预转染m i R -21抑制剂可抑制MV s 诱导的细胞肥大效应㊂结论 肾小管上皮细胞损伤时分泌包含m i R -21的MV s 可诱导心肌细胞的肥大,其可能参与了慢性肾脏病并发心力衰竭的进展㊂关键词:肾疾病;m i c r o R N A ;微囊泡;心肌肥大中图分类号:R 692 文献标志码:A 文章编号:1004-583X (2020)03-0245-06d o i :10.3969/j.i s s n .1004-583X.2020.03.011M i c r o v e s i c l e s c o n t a i n i n g m i r o r n a -21f r o mt u b u l a r e p i t h e l i a l c e l l s p r o m o t e c a r d i o m y o c y t e h y p e r t r o p h yD i J i a 1a ,Z h o uH u a 1a ,L iM i n 1a ,X uT i n g 1b ,Z h a o J i a b i 21a .D e p a r t m e n t o f N e p h r o l o g y ;b .D e p a r t m e n t o f R h e u m a t o l o g y a n dI mm u n o l o g y ,t h eT h i r dA f f i l i a t e d H o s pi t a l o f S o o c h o w U n i v e r s i t y ,C h a n g z h o u 213003,C h i n a ;2.D e p a r t m e n t o f P a t h o l o g y ,C h a n gz h o uS e c o n d P e o p l e 'sH o s p i t a l ,N a n j i n g M e d i c a lU n i v e r s i t y ,C h a n gz h o u 213003,C h i n a C o r r e s p o n d i n g a u t h o r :Z h a oJ i a b i ,E m a i l :z h a o ji a b i 1981@163.c o m A B S T R A C T :O b je c t i v e T o i n v e s t i g a t e t h e ef f e c t a n dm e c h a n i s mo fm i c r o v e s i c l e s (MV s )c o n t a i n i ng m i r o R N A -21(m i R -21)f r o m t u b u l a re p i th e li a lc e l l si nc a r d i o m y o c y t eh y p e r t r o p h y .M e t h o d s R e n a l t u b u l a re pi t h e l i a lc e l l s w e r e s t i m u l a t e db y t r a n s f o r m i n gg r o w t hf a c t o r β1(T G F -β1),a n dt h ec o n d i t i o n e d m e d i u m w a se x t r a c t e db y d i f f e r e n t i a l c e n t r i f u g a t i o n .R e n a lt u b u l a re p i t h e l i a lc e l l s w e r el a b e l e d w i t h D i l -C 18d y ea n dt h er e c i p i e n tc a r d i o m y o c y t e s w e r e o b s e r v e db y f l u o r e s c e n c e m i c r o s c o p e .M i R -21l e v e l i n MV sw a sd e t e c t e db yq P C R ,a n dt h e l e n g t ha n dd i a m e t e ro f c a r d i o m y o c y t e sw e r em e a s u r e db y e y e p i e c e .B i c i n c h o n i n i ca c i d (B C A )p r o t e i nk i t a n da t r i a l n a t r i u r e t i c p e p t i d e (A N P )k i tw e r e u s e d t o d e t e c t t h e c o n t e n t o f c e l l p r o t e i n a n d t h e l e v e l o fA N P .M i R -21i n h i b i t o rw a s t r a n s f e c t e d i n t o r e c i pi e n t c a r d i o m y o c y t e s i na d v a n c et oo b s e r v et h ee f f e c t so nt h e l e n g t ha n dd i a m e t e r ,p r o t e i nc o n t e n ta n d A N Pl e v e l o f t h e c e l l s .R e s u l t s T G F -β1c o u l d i n d u c e d o n o r r e n a l t u b u l a r e p i t h e l i a l c e l l s t o p r o d u c eMV sw h i c hw e r e t h e nd e l i v e r e d i n t o c a r d i o m y o c y t e s ,a n dt h ed i a m e t e r ,p r o t e i nc o n t e n ta n d A N Pl e v e lo fc a r d i o m y o c y t e s w e r es i g n i f i c a n t l y i n c r e a s e d .M e a n w h i l e ,m i R -21i n MV s c o u l d c a u s er e c i p i e n tc a r d i o m y o c y t e h y p e r t r o p h y .A n d p r e -t r a n s f e c t i o n o f m i R -21i n h i b i t o r s c o u l di n h i b i t MV s -i n d u c e dc a r d i o m y o c y t eh y p e r t r o p h y .C o n c l u s i o n T h ed a m a g e dr e n a l t u b u l a re p i t h e l i a l c e l l s c a n s e c r e t eMV s c o n t a i n i n g m i R -21w h i c h c a n i n d u c e c a r d i o m y o c y t e h y p e r t r o p h y .A n d t h i sm a y b e i n v o l v e d i n t h e p r o g r e s s o f c h r o n i ck i d n e y d i s e a s e c o m p l i c a t e dw i t hh e a r t f a i l u r e .K E Y W O R D S :k i d n e y d i s e a s e s ;m i c r o R N A ;m i c r o v e s i c l e s ;c a r d i o m y o c y t eh y p e r t r o p h y心血管疾病(C V D )是影响慢性肾脏病(C K D )患者预后最重要的因素,C K D 患者心血管死亡率约占总死亡率的44%[1],其中慢性心力衰竭为主要死因之一㊂心肌重构是心力衰竭发展的一个重要病理机制,目前认为抑制心肌重构是预防和治疗心力衰竭的重要手段,而心肌细胞肥大是心力衰竭发展过程中心肌重构的一个主要特征之一㊂m i c r o R N A(m i R N A )在心肌细胞肥大和凋亡中起到重要作用,其参与心力衰竭的发生和发展,并可作为心力衰竭㊃542㊃‘临床荟萃“ 2020年3月20日第35卷第3期 C l i n i c a l F o c u s ,M a r c h20,2020,V o l 35,N o .3Copyright ©博看网. All Rights Reserved.诊断㊁治疗和预后的新的生物学标志物及治疗靶点[2]㊂近年来,一种被称为 MV s 的细胞间通讯方式越来越受到研究者们的关注[3-4]㊂2007年,V a l a d i 发现微囊泡(MV s)中含有m i R N A㊂生理和病理状态下最基础的细胞代谢㊁增殖㊁分化和死亡等过程均受到m i R N A的调节[5],包含m i R N A的MV s作为重要的细胞间通讯的分子平台在疾病中的作用正成为新的研究前沿㊂心肌肥大时心脏m i R N A-21 (m i R-21)表达明显增多,有趣的是这些m i R-21大多数包含于MV s,存在于心脏周围液体中[6],这些MV s的来源尚不清楚㊂本文观察了肾小管上皮细胞产生的包含m i R-21的微囊泡在心肌肥大中的作用,从而可能拓展对疾病机制的理解,并可能为C K D导致心力衰竭的治疗新策略提供实验依据和理论基础㊂1材料与方法1.1细胞培养和处理大鼠近端肾小管上皮细胞系(N R K-52E)和大鼠心肌细胞(H9C2)购自美国模式菌种收集中心(A T C C)㊂用含10%胎牛血清(F B S,L i f e T e c h n o l o g y)和100U/m l青-链霉素(L i f e T e c h n o l o g y)的D-M E M/F12培养液(L i f e T e c h n o l o g y),在37ħ,含5%C O2的孵箱中培养细胞㊂细胞贴壁生长达到80%融合后,换成无血清的D-M E M/F12培养液孵育过夜(16h),再次更换无血清的D-M E M/F12培养液,加入重组人转化生长因子β1(r h T G F-β1,R&DS y s t e m s)处理,换成无血清的D-M E M/F12培养液再孵育细胞48h,收集此无血清㊁无r h T G F-β1的D-M E M/F12培养液,依次在4ħ的环境下离心:300gˑ5m i n,1200gˑ20m i n, 10000gˑ30m i n,弃沉淀,再经用滤器浓缩上清液,即为条件培养液㊂对照培养液的收集方法同上,但细胞未经T G F-β1处理㊂受体心肌细胞用条件培养液处理并检测㊂用脂质体转染试剂(L i f e T e c h n o l o g y)将m i R-21的抑制物或阴性对照(均购自Q i a g e n)转染H9C2㊂依据试剂说明书,细胞50%融合后,换成无抗生素㊁含10%胎牛血清(F B S)的D-M E M/F12培养液将适量m i R-21抑制物或阴性对照与250μl无血清培养基(L i f eT e c h n o l o g y)混合,室温5m i n,5μl脂质体转染试剂与250μl无血清培养基混合,室温20m i n,在37ħ,含5%C O2的孵箱中培养细胞6h后,更换为含抗生素㊁含10%F B S的培养液培养并给予相应的处理㊂1.2 MV s的提取和观察采用差速离心的方法提取条件培养液中的MV s,方法概述如下:收集细胞培养液依次在4ħ的环境下离心,300gˑ5m i n,1200gˑ20m i n,10000gˑ30m i n,弃沉淀,收集上清液在4ħ的环境下离心110000gˑ60m i n,沉淀物即为MV s㊂用适量的P B S重悬MV s㊂用T R I z o l®L S试剂(L i f eT e c h n o l o g y)提取MV s中的总R N A㊂用透射电镜观察MV s的形态,荧光染色观察MV s,方法参照文献[7],概括如下:D i l-C18是一种膜的荧光染料,用D i l-C18染料预处理供体N R K-52E细胞1h,用磷酸缓冲淮(P B S)清洗细胞3次,依据前文所述方法收集条件培养基,并离心得到被D i l-C18染色的MV s㊂对照MV s的收集方法类似,将MV s重悬在D-M E M/F12培养液中,孵育受体心肌细胞,用荧光显微镜观察受体细胞并拍照㊂1.3 m i R N A的实时定量P C R(q P C R)用T R I z o l (L i f eT e c h n o l o g y)提取组织或细胞中的总R N A,用m i S c r i p tR TI I试剂盒(Q i a g e n),以1μg的总R N A 为模板合成m i R N A的c D N A文库,反应体系为20μl(其中总R N A模板2μl),充分混匀,37ħˑ60 m i n,95ħˑ5m i n,立即置于冰上冷却,-20ħ保存㊂用m i S c r i p t S Y B RG r e e nP C R试剂盒Q i a g e n),以10n g的m i R N A的c D N A为模板扩增,反应体系为20μl(其中10ˑm i S c r i p tm i R-21或U6引物2μl, m i R N A的c D N A模板1μl),充分混匀,95ħˑ15 m i nˑ1次,(94ħˑ15s,55ħˑ30s,70ħˑ34s)ˑ40次,在70ħˑ34s步骤收集荧光信号㊂每个样本重复3次,采用A B I7300默认的阈值读取C T值, 3次平均值得到C T样本,ΔC T=C T m i R-21-C T U6,ΔΔC T=ΔC T处理-ΔC T对照,取2-ΔΔC T作m i R N A相对定量的柱状图㊂1.4心肌细胞长径的测量 H9C2心肌细胞消化后,培养基重悬细胞并进行计数,分别取2ˑ105个细胞到6孔板中,待细胞培养24h贴壁后更换细胞培养基为含2%F B S的D M E M培养基继续培养24h使细胞处于静止期㊂不同条件培养结束后,用目镜测微尺测量心肌细胞长径㊂400倍镜下每组随机选取6个胞浆横纹清晰㊁细胞核完整㊁形态规则的心肌细胞,测量细胞长径㊂1.5二喹啉甲酸(B C A)蛋白定量的检测 H9C2心肌细胞每孔分别加入100μl蛋白裂解液冰浴下对细胞进行充分裂解,细胞裂解后,收集裂解液于1.5m l 离心管中,12000g离心取上清,后按照B C A蛋白试剂盒说明进行定量㊂1.6酶联免疫吸附检测人心内肽(A N P)水平H9C2心肌细胞不同条件培养后,吸去H9C2心肌细胞的培养液,P B S清洗2遍后胰酶消化细胞,800g 离心收集细胞,200μl P B S重悬细胞并转移到新的1.5㊃642㊃‘临床荟萃“2020年3月20日第35卷第3期 C l i n i c a l F o c u s,M a r c h20,2020,V o l35,N o.3Copyright©博看网. All Rights Reserved.m l 离心管中㊂将离心管置于冰浴中超声(45W ,15s超声3次,间隔15s )以充分裂解㊂按照C U S A B I O 公司A N P 试剂盒的检测方法测定A N P 浓度㊂每个试验条件设3复孔同时进行实验,以其均值作为统计数据㊂1.7 统计学方法 用S i gm a S t a t 软件(版本3.5,J a n d e l S c i e n t i f i cS o f t w a r e )分析实验数据㊂组间比较采用方差分析和t 检验,P <0.05为差异有统计学意义㊂2 结 果2.1 T G F -β1可诱导供体肾小管上皮细胞产生MV s 以体外培养的N R K -52E 为研究对象,收集对照培养液及5n g /m l 的T G F -β1处理细胞24h 后的条件培养液,分别超速离心,留取沉淀用透射电镜观察,发现条件培养液沉淀物中有呈簇状的双层膜结构的小囊泡,直径在50~200n m ,符合MV s 的形态学特征,见图1㊂图1 肾小管上皮细胞产生的条件培养液超速离心获得的沉淀物的冷冻电镜照片 a .无T G F -β1处理的供体肾小管上皮细胞产生的条件培养液超速离心获得的沉淀物;b .5n g /m l 的T G F -β1处理的供体肾小管上皮细胞产生的条件培养液超速离心获得的沉淀物㊂标尺=100n m 2.2 D i l -C 18染料标记的MV s 可进入受体心肌细胞 用D i l -C 18染料标记培养的肾小管上皮细胞并收集T G F -β1处理后的条件培养液,超速离心获得D i l -C 18染料标记的MV s ,加入受体心肌细胞培养液中,并于不同时间点观察,受体心肌细胞内可见D i l -C 18红色荧光阳性的小颗粒,且数量随着处理时间的延长而增加,提示MV s 能从T G F -β1处理的肾小管上皮细胞传递到受体心肌细胞中,见图2㊂2.3 T G F -β1诱导产生的MV s 可促使受体心肌细胞肥大 为探讨MV s 对心肌细胞的作用,我们分别用条件培养液经超速离心后的上清液和沉淀的MV s 处理培养的心肌细胞,200μl 条件培养液沉淀的MV s 处理心肌细胞48h ,能导致细胞长径㊁蛋白浓度及A N P 含量的增加,而上清液没有上述效应㊂因此,结果提示T G F -β1处理后的供体肾小管上皮细胞分泌的MV s 能促使受体心肌细胞发生肥大效应,见图3㊂2.4 T G F -β1诱导肾小管上皮细胞分泌的M V s 中包含m i R -21 为探讨肾小管上皮细胞产生M V s 促使心肌细胞肥大的机制,我们收集了对照组和5n g /m l 的T G F -β1处理后的肾小管上皮细胞的培养液,超速离心分离条件培养液沉淀的MV s 后,分别提取总R N A 并检测其中的m i R -21含量,q P C R 分析显示,与对照组相比,T G F -β1处理的供体肾小管上皮细胞分泌的MV s 中m i R -21的含量显著增高㊂在此基础上,我们进一步确定MV s 能否将外源性m i R -21导入受体心肌细胞㊂同样,与含有m i R -21的供体N R K -52E 细胞条件培养液中分离的MV s 作用24h 后,受体心肌细胞中m i R -21的含量较对照组显著增加,见图4㊂2.5 包含m i R -21的MV s 诱导心肌细胞肥大 为进一步探讨外源性m i R -21在促进心肌细胞肥大中的作用,我们将m i R -21抑制剂预转染受体心肌细胞,并将收集的含有MV s 的条件培养液继续诱导心肌细胞48h ㊂转染m i R -21抑制剂的心肌细胞与转染N C 的对照组相比,条件培养液诱导组细胞长径㊁蛋白浓度和A N P 含量均明显增加,而预转染m i R -21抑制剂的受体心肌细胞则未发生肥大效应㊂这些结果表明,MV s 诱导的心肌细胞肥大可被m i R -21抑制剂所消除,见图5㊂㊃742㊃‘临床荟萃“ 2020年3月20日第35卷第3期 C l i n i c a l F o c u s ,M a r c h20,2020,V o l 35,N o .3Copyright ©博看网. All Rights Reserved.图2 用D i l -C 18染料标记M V s ,处理受体H 9C 2细胞不同时间后的荧光显微镜照片 a -d .供体N R K -52E 细胞经D i l -C 18染料标记,超速离心获得条件培养液的MV s ,处理受体N R K -52E 细胞0h (a )㊁6h (b )㊁12h (c )㊁24h (d )㊂红色:D i l -C 18标记的MV s ,蓝色:细胞核图3 200μl 条件培养液沉淀的M V s 处理心肌细胞48h 后细胞肥大效应 分别用条件培养液经超速离心后的上清液和沉淀的MV s 处理培养的心肌细胞细胞长径(a )㊁蛋白浓度(b )及A N P 含量(c )㊂*P <0.5图4 肾小管上皮细胞分泌的微囊泡中及受体心肌细胞中m i R -21的含量 a .对照培养液和条件培养液超速离心分离的MV s 中m i R -21的q -P C R 结果;b .对照培养液及条件培养液中分离的MV s 作用受体心肌细胞24h 后m i R -21的q -P C R 结果㊂*P <0.05㊃842㊃‘临床荟萃“ 2020年3月20日第35卷第3期 C l i n i c a l F o c u s ,M a r c h20,2020,V o l 35,N o .3Copyright ©博看网. All Rights Reserved.图5 M i R -21抑制剂预转染受体心肌细胞后用含有M V s 的条件培养液诱导48h 后细胞的肥大效应 分别用转染对照㊁含有MV s 的条件培养液㊁转染m i R -21抑制剂㊁含有MV s 的条件培养液+转染m i R -21抑制剂处理后的心肌细胞长径(a )㊁蛋白浓度(b )和A N P 含量(c )㊂*P <0.053 讨 论心力衰竭是C K D 患者死亡的重要原因,并发心力衰竭的C K D 患者数量呈持续增长,而C K D 患者普遍存在多种并发症,如高血压㊁糖尿病和动脉粥样硬化等,其均可独立地增加心脏风险,因此很难完全明确C K D 介导心力衰竭的潜在机制㊂肾素-血管紧张素-醛固酮系统(R A A S )抑制剂有逆转左心室肥厚,改善左心室功能的作用[8],但可能导致肾小球滤过率下降及血肌酐水平上升,引起高钾血症的危险性增加,限制了其在严重肾功能不全中的应用㊂而即使积极采取多种治疗手段如调整血流动力学㊁减轻液体超负荷㊁纠正贫血㊁营养不良,纠正骨矿物质代谢紊乱乃至透析治疗,仍不能完全逆转C K D 时心力衰竭的发生发展,提示肾-心交互作用的复杂性,需要积极探索其他心㊁肾相互作用的调控因子㊂细胞通讯是心㊁肾之间相互影响的重要方式㊂研究发现MV s 是介导非接触的细胞间信息通讯的重要载体[9]㊂本研究通过观察肾小管上皮细胞产生的MV s 在心肌肥大中的作用,为临床提供新的策略及手段㊂细胞在正常生理条件下可以产生少量MV s,在某些病理生理情况下产生明显增多[10]㊂本实验中我们首先用T G F -β1诱导肾小管上皮细胞损伤并收集这些细胞的条件培养液,采用低温超速离心方法观察到离心后获得的沉淀物在电镜下符合MV s 的形态学特征,且较正常对照明显增多㊂MV s 是细胞状态的缩影,一般情况下,细胞膜磷脂如磷脂酰丝氨酸(p h o s p h a t i d y l s e r i n e ,P S )和磷脂酰乙醇胺(p h o s p h a t i d y l e t h a n o l a m i n e ,P E )位于细胞膜内侧,当细胞内钙离子浓度升高时,P S 从细胞膜内侧翻向外侧[11]㊂本研究中我们用荧光染料D i l -C 18标记供体细胞,因D i l -C 18是一种脂质染料,可用于标记供体细胞的细胞膜,MV s 的脂质膜结构中通常包含部分来源细胞的细胞膜成分,故当细胞分泌MV s 时MV s 也被D i l -C 18标记,从而可用来验证供体细胞产生的MV s 能否进入受体细胞[12]㊂我们用供体细胞产生标记有D i l -C 18的MV s 处理受体心肌细胞,结果显示MV s 可以从肾小管上皮细胞传递到心肌细胞,并且处理时间越长,进入细胞的MV s 数目越多㊂为进一步验证这些MV s 的作用,我们用T G F -β1处理肾小管细胞,将收集的培养液经超速离心得到的上清液和沉淀的MV s 分别作用于心肌细胞,结果证实沉淀中的MV s 能够诱导心肌细胞肥大,而不含有MV s 的上清液则没有该效应,提示T G F -β1处理肾小管细胞产生的MV s 才是真正诱导心肌细胞肥大的原因㊂虽然MV s 是介导细胞间通讯的重要载体,然而真正介导细胞间作用的是MV s 中包含的各类分子㊂循环m i R N A 由于受到MV s 的保护非常稳定,避免了核糖核酸酶(R N a s e)的降解,作为运输m i R N A 的主要载体受到广泛关注[13]㊂其中,m i R -21能促使病理性心脏重塑和血管生成,成为心肌肥大作用中的研究热点[14]㊂有研究发现m i R -21在高血压肾损害中呈高表达,并推进高血压及其相关肾损害的病理进程[15],更提示外泌性MV s 在心肌肥大中的重要作用㊂在参与肾纤维化的多种m i R N A s 中,m i R -21也被证实起着十分重要的作用[16]㊂已证实纤维化肾脏小鼠模型中肾脏来源的m i R -21明显上调,且研究发现血循环中m i R -21的水平与肾纤维化程度亦呈正相关[17],提示肾脏纤维化时肾脏细胞分泌的m i R -21可能通过MV s 进入血循环㊂因此在慢性心㊁肾疾病的进展中,m i R -21均发挥着重要的调节作用㊂我们观察到用T G F -β1处理肾小管细胞产生的MV s 中含有大量的m i R -21,且这些MV s 作用于受体心肌细胞后可使心肌细胞中m i R -21含量显著增加,而转染m i R -21的抑制剂下调肾小管上皮细胞中m i R -21含量后,能够消除其导致的心肌肥大效㊃942㊃‘临床荟萃“ 2020年3月20日第35卷第3期 C l i n i c a l F o c u s ,M a r c h20,2020,V o l 35,N o .3Copyright ©博看网. All Rights Reserved.应,进一步验证了肾脏来源的包含m i R N A-21的MV s可作用于心肌细胞导致心肌肥大㊂综上所述,本研究发现T G F-β1处理后损伤的肾小管上皮细胞能够分泌包含m i R-21的MV s,并可介导m i R-21从肾小管上皮细胞传递到心肌细胞并引起心肌细胞的肥大㊂系统研究包含m i R N A的MV s 在心㊁肾细胞间的通讯作用及对靶基因的调控将进一步拓展对疾病机制的理解,并可能为C K D导致心力衰竭的治疗新策略提供实验依据和理论基础㊂参考文献:[1] T a n a k aK,W a t a n a b eT,T a k e u c h iA,e ta l.C a r d i o v a s c u l a re v e n t sa n d d e a t hi n J a p a n e s e p a t i e n t s w i t h c h r o n i c k i d n e yd i se a s e[J].K i d n e y I n t,2017,91(1):227-234.[2]徐曼曼,张久亮,范书英,等.M i c r o R N A s与心力衰竭的研究进展[J].中国全科医学,2018,21(9):1030-1035.[3] L a t i f k a rA,H u rY H,S a n c h e zJ C,e ta l.N e wi n s i g h t s i n t oe x t r a c e l l u l a rv e s i c l eb i o g e n e s i sa n df u n c t i o n[J].JC e l lS c i,2019,132(13)p i i:j c s222406.[4]刘中,产进中,张野,等.外泌体微小R N A在心血管疾病中作用的研究进展[J].中华实用诊断与治疗杂志,2018,32(9): 934-936.[5] A m b r o sV.T h e f u n c t i o n s o f a n i m a lm i c r o R N A s[J].N a t u r e,2004,431(7006):350-355.[6] B a n g C,B a t k a i S,D a n g w a l S,e t a l.C a r d i a c f i b r o b l a s t-d e r i v e dm i c r o r n a p a s s e n g e r s t r a n d-e n r i c h e d e x o s o m e s m e d i a t ec a rd i o m y o c y t eh y pe r t r o p h y[J].JC l i nI n v e s t,2014,124(5):2136-2146.[7] Z h a n g Y,L i uD,C h e nX,e t a l.S e c r e t e dm o n o c y t i cm i R-150e n h a n c e st a r g e t e d e n d o t h e l i a lc e l l m i g r a t i o n[J].M o l e c u l a rC e l l,2010,39(1):133-144.[8] O n u i g b oMA.R A A S i n h i b i t i o na n dc a r d i o r e n a l s y n d r o m e[J].C u r rH y p e r t e n sR e v,2014,10(2):107-111.[9] V a l a d iH,E k s t r o m K,B o s s i o sA,e ta l.E x o s o m e-m e d i a t e dt r a n s f e r o fm R N A sa n d m i c r o R N A s i san o v e lm e c h a n i s m o fg e n e t i c e x c h a n g e b e t w e e n c e l l s[J].N a t C e l l B i o l,2007,9(6):654-659.[10] B o r g e s F T,M e l o S A,O z d e m i r B C,e t a l.T G F-b e t a1-c o n t a i n i n g e x o s o m e s f r o m i n j u r ede p i t h e l i a l c e l l s a c t i v a t ef i b r o b l a s t s t o i n i t i a t e t i s s u e r eg e n e r a t i v e r e s p o n s e s a n d f i b r o s i s[J].JA mS o cN e p h r o l,2013,24(3):385-392. [11] H u g e lB,M a r t i n e z M C,K u n z e l m a n n C,e ta l.M e m b r a n em i c r o p a r t i c l e s:t w o s i d e s o f t h e c o i n[J].P h y s i o l o g y(B e t h e s d a),2005,20(1):22-27.[12] Z h o u Y,X i o n g M,F a n g L,e t a l.m i R-21-c o n t a i n i n gm i c r o v e s i c l e sf r o m i n j u r e d t u b u l a r e p i t h e l i a lc e l l s p r o m o t e t u b u l a r p h e n o t y p et r a n s i t i o nb y t a r g e t i n g P T E N p r o t e i n[J].A mJP a t h o l,2013,183(4):1183-1196.[13] M e l a k T,B a y n e s HW.C i r c u l a t i n g m i c r o R N A sa s p o s s i b l eb i o m a r k e r s f o rc o r o n a r y a r t e r yd i se a s e:a n a r r a t i v e r e v i e w[J].E J IF C C,2019,30(2):179-194.[14] Y u a nJ,C h e n H,G e D,e ta l.M i r-21p r o m o t e sc a r d i a cf i b r o s i s a f t e r m y o c a r d i a l i n f a r c t i o nv i at a rg e t i n g s m a d7[J].C e l l P h y s i o l B i o c h e m,2017,42(6):2207-2219.[15]韩聪,姜月华,李伟,等.m i R N A-21在高血压肾损害中的作用机制及研究进展[J].中华高血压杂志,2019,27(8):728-733.[16] C h u p p aS,L i a n g M,L i u P,e ta l.M i c r o R N A-21r e g u l a t e sp e r o x i s o m e p r o l i f e r a t o r-a c t i v a t e dr e c e p t o ra l p h a,a m o l e c u l a rm e c h a n i s mo f c a r d i a c p a t h o l o g y i n c a r d i o r e n a l s y n d r o m e t y p e4[J].K i d n e y I n t,2018,93(2):375-389.[17] H eY,H u a n g C,L i J.M i R-21i s a c r i t i c a l t h e r a p e u t i c t a r g e tf o r r e n a l f i b r o s i s[J].C e l lB i o c h e m B i o p h y s,2014,68(3):635-636.收稿日期:2019-12-05编辑:张卫国㊃052㊃‘临床荟萃“2020年3月20日第35卷第3期 C l i n i c a l F o c u s,M a r c h20,2020,V o l35,N o.3Copyright©博看网. All Rights Reserved.。
高良姜素减轻异丙肾上腺素诱导心脏纤维化的作用机制

高良姜素减轻异丙肾上腺素诱导心脏纤维化的作用机制尚茹茹1,童曼琳2,刘晓红1摘要目的:探讨高良姜素(Galangin)对异丙肾上腺素(ISO)诱导心脏纤维化的作用机制㊂方法:将30只C57BL/6小鼠随机分为正常组㊁ISO组㊁高良姜素低剂量组[25mg/(kg㊃d)]㊁高良姜素中剂量组[50mg/(kg㊃d)]㊁高良姜素高剂量组[100mg/(kg㊃d)]㊂处理结束后行心脏超声评估心功能[左室射血分数(LVEF)㊁左室短轴缩短率(LVFS)㊁左室收缩末期内径(LVESD)㊁左室舒张末期内径(LVEDD)];测量小鼠体质量(BW)㊁心脏重量(HW)㊁胫骨长度(TL),计算心脏重量与体质量比值(HW/BW)㊁心脏重量与胫骨长度比值(HW/TL),评估心肌肥厚情况㊂天狼星红染色评价心脏纤维化程度;实时荧光定量聚合酶链式反应(qPCR)检测炎症标志物白细胞介素-6(IL-6)㊁白细胞介素-1β(IL-1β)㊁肿瘤坏死因子-α(TNF-α)和纤维化标志物胶原蛋白Ⅰ(CollagenⅠ)㊁胶原蛋白Ⅲ(CollagenⅢ)㊁转化生长因子-β1(TGF-β1)mRNA水平;试剂盒检测心脏组织氧化应激指标超氧化物歧化酶(SOD)㊁过氧化氢酶(CAT)活性和丙二醛(MDA)含量㊂结果:与正常组比较,ISO组小鼠HW/BW㊁HW/TL㊁LVEDD及LVESD升高,LVEF和LVFS降低,IL-6㊁IL-1β㊁TNF-α㊁CollagenⅠ㊁CollagenⅢ和TGF-β1mRNA表达水平升高,心脏SOD和CAT活性降低,MDA含量升高,心肌细胞横截面积增大,心脏纤维化面积增加(P<0.05)㊂与ISO组比较,高良姜素高剂量组心脏纤维化面积减小,CollagenⅠ㊁CollagenⅢ㊁IL-6mRNA表达水平降低,SOD活性升高,MDA含量降低,差异有统计学意义(P<0.05)㊂高良姜素中剂量组TGF-β1低于ISO组,差异有统计学意义(P<0.05)㊂结论:高良姜素可减轻ISO诱导的心脏纤维化,其机制可能与抑制炎症㊁氧化应激有关㊂关键词心脏纤维化;高良姜素;异丙肾上腺素;炎症;氧化应激;小鼠;实验研究d o i:10.12102/j.i s s n.1672-1349.2023.24.009Mechanism of Galangin on Isoprenaline-induced Cardiac FibrosisSHANG Ruru,TONG Manlin,LIU XiaohongShanxi Provincial People's Hospital,Taiyuan030012,Shanxi,ChinaCorresponding Author LIU Xiaohong,E-mail:****************Abstract Objective:To investigate the mechanism of Galangin on isoprenaline(ISO)-induced cardiac fibrosis.Methods:Thirty C57BL/6 mice were randomly divided into the normal group,ISO group,low-dose galangin group[25mg/(kg㊃d)],medium-dose galangin group [50mg/(kg㊃d)],and high-dose galangin group[100mg/(kg㊃d)].After treatment,cardiac ultrasound was performed to detect cardiac function[left ventricular ejection fraction(LVEF),left ventricular short-axis shortening(LVFS),left ventricular end-systolic internal diameter(LVESD),and left ventricular end-diastolic internal diameter(LVEDD)].The body mass(BW),heart weight(HW),and tibia length (TL)of the mice were measured,and the heart weight-weight to body mass ratio(HW/BW)was detected,and the heart weight to tibia length ratio(HW/TL)were collected to assess cardiac hypertrophy.The degree of cardiac fibrosis was detected by sirius scarlet staining.The inflammatory markers interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and fibrotic markers collagen Ⅰ(CollagenⅠ),collagenⅢ(CollagenⅢ),transforming growth factor-β1(TGF-β1)mRNA levels were detected by real-time fluorescence quantitative polymerase chain reaction(qPCR).The kit was used to detect the oxidative stress indicators superoxide dismutase(SOD), catalase(CAT)activity,and malondialdehyde(MDA)content in heart tissue.Results:Compared with the normal group,HW/BW,HW/TL, LVEDD,and LVESD in the ISO group were higher,LVEF and LVFS were less,levels of IL-6,IL-1β,TNF-α,CollagenⅠ,CollagenⅢ,and TGF-β1mRNA were higher,SOD and CAT activities were less,and MDA content was higher.The cardiomyocyte cross-sectional area and cardiac fibrosis area were higher(P<0.05).Compared with the ISO group,the area of cardiac fibrosis was less in the high-dose galangin group,the expression levels of CollagenⅠ,CollagenⅢ,and the mRNA of IL-6were less,the activity of SOD was higher,and the content of MDA was less(P<0.05).TGF-β1in the medium-dose galangin group was less than that in the ISO group(P<0.05). Conclusion:Galangin could alleviate isoproterenol-induced cardiac fibrosis,and its mechanism might be related to inhibiting inflammation and oxidative stress.Keywords cardiac fibrosis;Galangin;isoproterenol;inflammation;oxidative stress;mice;experimental study心力衰竭是心血管疾病的终末阶段,发病率及死基金项目山西省卫生健康委科研课题项目(No.2021112)作者单位 1.山西省人民医院(太原030012);2.山西医科大学第五临床医学院通讯作者刘晓红,E-mail:****************引用信息尚茹茹,童曼琳,刘晓红.高良姜素减轻异丙肾上腺素诱导心脏纤维化的作用机制[J].中西医结合心脑血管病杂志,2023,21(24): 4524-4529.亡率均较高,心力衰竭发生机制复杂,尚未明确㊂目前认为神经内分泌系统激活导致的心室重塑是引起心力衰竭的关键因素,心脏纤维化是心室重塑的主要病理特征,炎症在心脏纤维化过程中发挥着重要作用[1]㊂心力衰竭发展过程中产生大量的促炎因子,这些促炎因子导致心肌细胞肥大和心脏纤维化㊂因此,抑制心脏炎症㊁纤维化有助于改善心力衰竭预后㊂高良姜素(Galangin,3,5,7-三羟基黄酮)是一种天然来源的黄酮醇类化合物,主要分布于姜科植物高良姜的干燥根茎中㊂多项研究显示,高良姜素具有抗炎㊁抗氧化㊁抗凋亡㊁抗肿瘤及抗纤维化等作用[2-3]㊂有研究显示,高良姜素可减少活性氧生成,抑制丝裂原活化蛋白激酶(MAPK)信号通路,减轻哮喘小鼠气道炎症反应及气道重塑[4]㊂本研究探讨高良姜素通过抗炎㊁抗氧化㊁抗纤维化等抑制心脏纤维化发挥抗心力衰竭的作用机制㊂1材料与方法1.1实验材料1.1.1动物模型及分组C57BL/6小鼠购自北京斯贝福生物技术有限公司[生产许可证:SCK(京)2019-0010],6~8周龄,雄性,体质量23.5~25.5g㊂饲养于无特定病原体(SFP)级环境中,自由摄取食物,昼夜12h㊂异丙肾上腺素(ISO)组(6只)小鼠在实验第1日皮下注射ISO[5mg/(kg㊃d)],共治疗14d,之后每日灌胃对应体积的0.5%羟甲基纤维素溶液,持续4周㊂高良姜素各剂量组(每组6只)在实验第1天皮下注射ISO[5mg/(kg㊃d)]同时给予不同浓度的高良姜素(重悬于0.5%羟甲基纤维素溶液,每次灌胃前混匀)灌胃,ISO使用14d,之后持续灌胃高良姜素至4周㊂正常组(6只)从实验开始每日灌胃对应体积的0.5%羟甲基纤维素溶液,持续4周㊂详见图1㊂图1小鼠的处理方法(CMC为羧甲基纤维素;Gla为高良姜素)1.1.2实验试剂与仪器高良姜素(上海融禾医药科技发展有限公司); ISO(sigma,美国);异硫氰酸荧光素(FITC)标记的麦胚芽凝集素(WGA)染色试剂盒(Sigma,美国);天狼星红染色试剂盒(北京索莱宝);超氧化物歧化酶(SOD)试剂盒(南京建成);丙二醛(MDA)试剂盒(南京建成);过氧化氢酶(CAT)试剂盒(南京建成);心脏超声测量系统(美国GE公司Vivid7pro超声系统);荧光显微镜(Nikon);实时荧光定量聚合酶链式反应(qPCR)仪(Bio-Rad)㊂1.2方法1.2.1小鼠心脏超声心动图检查实验4周,行心脏彩超检测小鼠左室射血分数(LVEF)㊁左室短轴缩短率(LVFS)㊁左室收缩末期内径(LVESD)㊁左室舒张末期内径(LVEDD),上述数据测量3次,取平均值,记录分析㊂1.2.2小鼠体质量㊁心脏重量及胫骨长度检测完成心脏超声的小鼠,测量体质量(body weight, BW),每只小鼠称量3次,取平均值并记录㊂给予1%戊巴比妥钠溶液(20mg/kg)腹腔注射,前胸部开口,暴露心脏,心尖部穿刺,用肝素盐水灌注5min,取材㊂测量心脏重量(heart weight,HW)㊁胫骨长度(tibial length,TL)㊂计算心脏重量与体质量比值(HW/BW)㊁心脏重量与胫骨长度比值(HW/TL)㊂1.2.3组织取材㊁石蜡切片制备㊁常规染色组织取材后,经10%甲醛固定,乙醇脱水㊁石蜡包埋,垂直于心脏长轴连续切成5μm的石蜡组织切片,进行WGA染色㊂采用Nikon显微镜收集图像,并使用Image J软件进行定量分析㊂1.2.4qPCR检测提取小鼠心脏组织总RNA后,将2μg的RNA按照说明书反转录为cDNA㊂应用SYBR(Servicebio公司),按照说明书检测相关基因,引物序列如下:胶原蛋白(Collagen)Ⅰ,正向引物5'AAGAAGCACGTCTG-GTTTGGAG3',反向引物5'GGTCCATGTAGGC-TACGCTGTT3';CollagenⅢ,正向引物5'TTTCTTCT-CACCCTTCTTCATCC3',反向引物5'CATATTTGA-CATGGTTCTGGCTTC3';转化生长因子-β1(TGF-β1),正向引物5'TAATGGTGGACCGCAACAAC3',反向引物5'CCACATGTTGCTCCACACTTGAT3';白细胞介素-1β(IL-1β),正向引物5'TCAAATCTCGCAGCAG-CACATC3',反向引物5'CGTCACACACCAGCAGGT-TATC3';IL-6,正向引物5'CCCCAATTTCCAAT-GCTCTCC3';反向引物5'CGCACTAGGTTTGC-CGAGTA3';肿瘤坏死因子-α(TNF-α),正向引物5' CCCTCACACTCACAAACCACC3',反向引物5'CTTT-GAGA TCCA TGCCGTTG3';磷酸甘油醛脱氢酶(GAPDH),正向引物5'CCTCGTCCCGTAGACAAAATG3',反向引物5'TGAGGTCAATGAAGGGGTCGT3'㊂1.2.5心脏组织氧化应激水平检测取适量心脏组织,按照试剂盒说明书步骤分别进行SOD和CA T活性检测,同时检测心脏组织MDA含量㊂1.3统计学处理采用SPSS26.0统计软件对数据进行分析,符合正态分布的定量资料以均数ʃ标准差(xʃs)表示,组间比较采用独立样本t检验,多组间比较采用单因素方差分析㊂以P<0.05为差异有统计学意义㊂统计图表制作使用GraphPad Prism9.5软件完成㊂2结果2.1各组小鼠心脏组织形态学比较与正常组比较,ISO组小鼠HW/BW㊁HW/TL升高,差异有统计学意义(P<0.05)㊂详见图2㊂图2各组小鼠心脏组织形态学比较(A为HW/BW;B为HW/TL㊂与正常组比较,*P<0.05)2.2各组小鼠心肌细胞横截面积及心脏纤维化面积比较WGA染色结果显示:与正常组比较,ISO组小鼠心肌细胞横截面积增大(P<0.05)㊂天狼星红染色结果显示,与正常组比较,ISO组小鼠心脏纤维化程度加重(P<0.05);与ISO组比较,高良姜素高剂量组心脏纤维化程度减轻(P<0.05)㊂详见图3㊁图4㊂图3各组小鼠心肌细胞横截面积及心脏纤维化染色图(ˑ400) (A为WGA染色代表图;B为天狼星红染色代表图)图4各组小鼠心肌细胞横截面积及心脏纤维化面积比较柱状图(A为心肌细胞横截面积;B为心脏纤维化面积㊂与正常组比较,*P<0.05;与ISO组比较,#P<0.05)2.3各组小鼠心功能指标比较与正常组比较,ISO组小鼠LVEF和LVFS下降,LVEDD㊁LVESD升高,差异有统计学意义(P<0.05)㊂详见图5㊂图5各组小鼠心功能指标比较的柱状图(A为LVEF;B为LVFS;C为LVEDD;D为LVESD㊂与正常组比较,*P<0.05)2.4各组小鼠心脏CollagenⅠ㊁CollagenⅢ㊁TGF-β1 mRNA表达水平比较与正常组比较,ISO组小鼠心脏纤维化标志物CollagenⅠ㊁CollagenⅢ㊁TGF-β1mRNA表达水平升高(P<0.05)㊂与ISO组比较,高良姜素高剂量组心脏纤维化标志物CollagenⅠ㊁CollagenⅢmRNA水平降低,高良姜素中剂量组心脏纤维化标志物TGF-β1 mRNA表达水平降低,差异有统计学意义(P<0.05)㊂详见图6㊂图6各组小鼠心脏CollagenⅠ㊁CollagenⅢ㊁TGF-β1mRNA表达水平比较的柱状图(A为CollagenⅠmRNA;B为CollagenⅢmRNA表达水平;C为TGF-β1mRNA表达水平㊂与正常组比较,*P<0.05;与ISO组比较,#P<0.05)2.5各组小鼠心脏组织IL-6㊁IL-1β㊁TNF-αmRNA表达水平比较与正常组比较,ISO组心脏炎症标志物IL-6㊁IL-1β㊁TNF-αmRNA表达水平升高(P<0.05)㊂与ISO组比较,高良姜素高剂量组心脏炎症标志物IL-6mRNA降低,差异有统计学意义(P<0.05)㊂详见图7㊂图7各组小鼠心脏组织IL-6㊁IL-1β㊁TNF-αmRNA表达水平比较的柱状图(A为IL-6mRNA表达水平;B为IL-1βmRNA表达水平;C为TNF-αmRNA表达水平㊂与正常组比较,*P<0.05;与ISO组比较,#P<0.05)2.6各组小鼠心脏组织氧化应激指标SOD㊁MDA㊁CAT水平比较与正常组比较,ISO组小鼠心脏组织SOD㊁CAT 活性降低,心脏组织MDA含量升高,差异有统计学意义(P<0.05)㊂与ISO组比较,高良姜素高剂量组SOD活性升高,MDA含量下降,差异有统计学意义(P< 0.05)㊂详见图8㊂图8各组小鼠心脏组织氧化应激指标SOD㊁MDA㊁CAT水平比较的柱状图(A为心脏组织SOD活性;B为心脏组织CAT的活性;C为心脏组织MDA含量㊂与正常组比较,*P<0.05;与ISO组比较,#P<0.05)3讨论心室重塑是心力衰竭发生发展中的重要病理过程,心肌细胞在持续压力超负荷㊁缺血㊁缺氧㊁炎症等应激条件下发生结构和功能改变,包括左心室形态改变㊁心肌细胞肥大和间质纤维化㊁心肌细胞凋亡[5]㊂心肌损伤的早期阶段,大量的炎症细胞浸润到心肌损伤区域并发挥作用㊂促炎信号通路是由多种促炎细胞因子(如TNF-α㊁IL-17㊁IL-1β㊁IL-6)介导的,可诱导心肌细胞肥大和凋亡,进一步加重炎症和纤维化,最终导致心室重塑[6]㊂氧化应激损伤在心脏纤维化过程中发挥着关键作用㊂病理情况下,活性氧过量积累导致脂质过氧化反应发生,促使细胞损伤和死亡,引起细胞功能障碍[7]㊂活性氧分子经抗氧化酶如CAT和SOD降解为无毒分子㊂抗氧化防御机制(SOD㊁CAT)或内源性抗氧化剂(维生素E㊁抗坏血酸和谷胱甘肽)浓度降低可增加活性氧水平㊂有研究显示,心力衰竭病人血浆和心包积液中脂质过氧化物水平升高,与病人严重程度呈正相关[7]㊂活性氧引发心脏成纤维细胞增殖,激活核因子-κB (nuclear factor kappa B,NF-κB),以此增加基质金属蛋白酶表达,导致细胞外基质聚集,促进心肌纤维化[8]㊂相关研究表明,竹叶提取物通过缓解氧化应激损伤,减轻糖尿病诱导的心脏纤维化[9]㊂有研究显示,顺铂诱导的小鼠肾损伤过程中,高良姜素通过抗炎㊁抗氧化应激及抗凋亡作用发挥肾脏保护作用[10];在链脲霉素诱导的糖尿病大鼠中,高良姜素通过改善肝脏的抗氧化应激水平,维持高血糖大鼠肝脏的线粒体功能[11];高良姜素通过抑制炎性因子表达,减轻尿酸诱导的肾脏炎症反应[12]㊂本研究结果显示,在ISO诱导的心脏损伤及纤维化模型中,高良姜素干预后降低了HW/BW㊁HW/TL,减轻了心肌间质纤维化程度,缩小了心肌细胞的横截面积,改善了心功能;高良姜素干预后下调了心脏炎症标志物(IL-6㊁IL-1β㊁TNF-α)和心脏纤维化标志物(CollagenⅠ㊁CollagenⅢ㊁TGF-β1)mRNA表达水平,同时升高了心脏组织中抗氧化酶SOD㊁CAT活性,降低了心脏组织中脂质氧化物MDA的含量㊂综上所述,高良姜素通过减轻心脏炎症反应㊁心脏氧化应激反应,抑制心脏成纤维细胞向肌成纤维细胞转化,从而减轻ISO诱导的心脏损伤及纤维化㊂但具体分子机制尚未明确,在今后的研究中将深入探讨㊂参考文献:[1]SHIRAZI L F,BISSETT J,ROMEO F,et al.Role of inflammation inheart failure[J].Current Atherosclerosis Reports,2017,19(6):27. [2]ABUKHALIL M H,ALTHUNIBAT O Y,ALADAILEH S H,et al.Galangin attenuates diabetic cardiomyopathy through modulatingoxidative stress,inflammation and apoptosis in rats[J].Biomedecine&Pharmacotherapie,2021,138:111410. [3]TANG X X,XU C M,YAGIZ Y,et al.Phytochemical profiles,andantimicrobial and antioxidant activities of greater galangal[Alpinia galanga(Linn.)Swartz]flowers[J].Food Chemistry,2018,255:300-308.[4]LIU Y N,ZHA W J,MA Y,et al.Galangin attenuates airwayremodelling by inhibiting TGF-β1-mediated ROS generation andMAPK/AKT phosphorylation in asthma[J].Scientific Reports,2015,5:11758.[5]GAO G Y,CHEN W W,YAN M J,et al.Rapamycin regulates thebalance between cardiomyocyte apoptosis and autophagy inchronic heart failure by inhibiting mTOR signaling[J].International Journal of Molecular Medicine,2020,45(1):195-209.[6]ADAMO L,ROCHA-RESENDE C,PRABHU S D,et al.Reappraising the role of inflammation in heart failure[J].NatureReviews Cardiology,2020,17(5):269-285.[7]TSUTSUI H,KINUGAWA S,MATSUSHIMA S.Oxidative stressand heart failure[J].American Journal of Physiology Heart andCirculatory Physiology,2011,301(6):H2181-H2190.[8]MARCHIOLI R,LEVANTESI G,MACCHIA A,et al.Vitamin Eincreases the risk of developing heart failure after myocardialinfarction:results from the GISSI-Prevenzione trial[J].Journal ofCardiovascular Medicine,2006,7(5):347-350.[9]ZHANG L L,MAO Y Z,PAN J J,et al.Bamboo leaf extractameliorates cardiac fibrosis possibly via alleviating inflammation,oxidative stress and apoptosis[J].Biomedicine&Pharmacotherapy,2017,95:808-817.[10]TOMAR A,VASISTH S,KHAN S I,et al.Galangin amelioratescisplatin induced nephrotoxicity in vivo by modulation ofoxidative stress,apoptosis and inflammation through interplay ofMAPK signaling cascade[J].Phytomedicine,2017,34:154-161.[11]ALOUD A A,VEERAMANI C,GOVINDASAMY C,et al.Galangin,anatural flavonoid reduces mitochondrial oxidative damage instreptozotocin-induced diabetic rats[J].Redox Report,2018,23(1):29-34.[12]LU H,YAO H,ZOU R,et al.Galangin suppresses renalinflammation via the inhibition of NF-κB,PI3K/AKT and NLRP3inuric acid treated NRK-52E tubular epithelial cells[J].Bio MedResearch International,2019,2019:1-10.(收稿日期:2023-03-17)(本文编辑薛妮)。
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Study on the Fatigue Properties of the TIG Weld Joint with the Stainless SteelConduit in AircraftYing Liu, Dongjie Li*, Xiaohong LiAvic Beijing Aeronautical Manufacturing Technology Research Institute,10024, China* Corresponding author: djli@Abstract The research focus on the material of the stainless steel thin conduit in aircraft, named 1Cr18Ni9Ti , and the TIG weld joint of which was investigated to analysis the fatigue properties. The fracture mechanics was used to analysis the crack initiation life and crack propagation life, and the fatigue surface was characterized with scanning electron microscope (SEM). The experimental and analytical results show that, the origin position of fatigue crack is the surface of the conduit. The stress concentration at the weld toe, the crystal structure is not uniform and Stress concentration in the heat affected zone (HAZ) and fusion line, so the fatigue cracks are easily generated in these locations. Delta K increases to a certain value, the HAZ has become one of the most dangerous position. The crack initiation life of HAZ in the total fatigue life is far higher than the proportion of crack propagation life.Keywords1Cr18Ni9Ti stainless steel, Fracture mechanics, Fatigue life, Fatigue crack1. IntroductionThe fatigue fracture is the major form of the metal structure failing, and which may be in the total percentage of 80%. The fatigue fracture of the welding structure is mainly because of the fatigue cumulative damage of the weld joint. Therefore, the fatigue performance of the weld joint directly affects the ability of anti fatigue fracture of weld structure[1].Aero-engine conduit system is an important part of the engine accessory system, which is mainly used for the transportation of fuel, hydraulic, lubricating oil and air. The conduit system is working under the complex stress state, contents the internal pressure of a liquid or gas, the assembly stress and the dynamic stress caused by engine vibration. Conduit state directly affects the safety of the engine, and the connecting method of the conduit is mainly used by the welding technology. According to reports, in the process of the development of new aircraft, defects in the weld zone (WZ) and the crack of the HAZ causing the conduit fracture or joint leakage fault is very frequent. As one type of aero-engine appeared continuously two fuel leak, causing the engine parking accident in the air. To analyze the accident indicated that, the relatively bigger assembly stress caused the conduit fatigue fracture, and in this process the welding defects play an important role in the promoting of the fatigue crack initiation.Aero-engine conduit material 1Cr18Ni9Ti belongs to the austenitic stainless steel, it has the characteristics of good corrosion resistance, flexibility, vibration resistance and wide temperature using range, at the same time, this steel has good mechanical properties and oxidation resistance, which is widely used in aircraft hydraulic, fuel, gas conduit system. In accordance with the conventional TIG welding of 1Cr18Ni9Ti weld joint of the typical of aero-engine conduit structure, the bending fatigue test was carried out to analysis the fatigue life of the welding structure. Fatigue crack growth rate testing of 1Cr18Ni9Ti TIG welding joint was carried out to analysis the crack initiation life and the crack propagation life. The SEM, optical microscope (OM) methods was used to observation and analysis the fatigue fracture.2. ExperimentalFigure 1. Bending fatigue specimenTest material is 1Cr18Ni9Ti austenitic stainless steel and the chemical composition is 0.10C, 1.56Mn, 0.70Si, 17.75Cr, 9.84Ni, 0.45Ti, the other is Fe. As service, the stress ratio of vibration fatigue load of the 1Cr18Ni9Ti conduit during is -1. Because the test object for the conduit is the thin wall structure, test of the vibration fatigue has certain difficulty. So the rotary bending fatigue of the weld joint was used to simulate the service load conditions. The TIG welding structure for the fatigue life test sample is shown in Fig. 1. 1Cr18Ni9Ti conduit and the processed butt was welded under the current 27 A, filled with diameter of 1 mm welding wire in the same chemical composition of base metal. The stress ratio R of the test is -1, the average str ess σm is 0, the stress peak value is between 200-300 MPa. For the butt welding of 10×1 mm conduit, the mechanical properties of weld joint is shown in table 1.Table 1. mechanical properties of base metal and weld jointR p0.2(MPa) R m(MPa) A(%)Base metal 196 600 66.5Weld joint 279 567 -Figure 2. Three point bending specimenThe fatigue crack growth rate tests using the same welding process welding as the conduit welding. Welding test plate is processed into a three point bend specimen standard, the thickness of the sample is 4 mm, and the sample shape is shown in Fig. 2. The test used the INSTRON 8801 50KN testing machine. The change of crack length obtained by the extensometer gap mouth displacement record, the calculation process is automatically completed by the testing machine. After the test, metallographic examination and fracture analysis of microstructure were performed in the OLYMBUS BX51M OM and CS-3400 type SEM.3. Results and discussion3.1. Analysis of fatigue life of welded conduitFigure 3. Fatigue test dataThe actual vibration stress value of the application welded conduit is less than 100 MPa, the purpose in this research of improving the stress peak value to 200-300 MPa is to shorten the test cycle. Fig. 3 are shown the 1Cr18Ni9Ti welding test results of fatigue life under the different stress, and from which we calculate the median fatigue life of N50 according to the original data. In double logarithmic coordinates, N50 data points into a linear distribution; linear fitting by least square method to the S-N relation is obtained:lgS=537.37253-52.9539lgN (1) From Eq. 1, when the peak stress in the 100 MPa, the fatigue life of welded conduit is 1.28×1010, which performance good fatigue property.Figure 4a. The SEM for the fracture morphologyFigure 4b. The SEM for the fatigue fracture zoneFigure 4c. The SEM for the ductile fracture zoneFigure 4. The SEM for the fracture morphologyFig. 4 is the bending fatigue specimen SEM photograph (Fig. 4A) and fracture detail picture (Fig. 4B and Fig. 4C). Fatigue fracture zone exist obvious bending fatigue specimen fracture (region I) and ductile fracture zone (II), as shown in Fig. 4a. Fatigue fracture zone detail pictures as shown in Fig. 4b, the fracture is with no obvious plastic deformation and the inter wall of the conduit can be observed fatigue crack source. The fracture exist obvious local tearing ridge, since the beginning of the specimen surface within a radial divergence state. The crack originated from the surface of the sample.Fig. 4C shows a ductile fracture zone picture. For ductile fracture zone, the fracture morphology is full of dimples. Combined with the Fig. 4a, the plastic deformation is obvious in this area, which is due to fatigue test loading way. This process is accompanied with torsion and shear stress, which increased the deformation of conduit joint.From fracture analysis of Fig. 4, fatigue crack originated in the catheter surface defects. The fatigue crack is on the one hand along the radial extension to the surface, on the other hand along the circumferential expansion. Continued expansion of cracks, the stress on the remaining section continues to increase, while the remaining area is not enough to support the load, i.e. instantaneousoccurrence of fracture[2-4].Figure 5. OM for the rotating bending fatigue specimenRotating bending fatigue specimen is shown in Fig. 5. From Fig. 5, the fracture position is the weld toe. Because the height position of weld fusion line is between the base metal and weld joint, the stress concentration lead the conditions for the generation of fatigue crack, reduces the fatigue life of welded joints.3.2. The crack initiation life and propagation lifeFigure 6a. OM for the crack of WZFigure 6b. OM for the crack of HAZFigure 6. OM for the crack of WZ and HAZFig. 6 shows the fracture of crack growth rate test (Fig. 6a) and heat affected zone (Fig. 6b) morphology of fracture surface. As shown in Fig. 6a, weld crack is perpendicular to the weld columnar crystal, and interface of the crack is smooth, showing uniform microstructure and residual stress state. As shown in Fig. 6B, the heat affected zone interface is undulating, from the heat affected zone extends to the fusion line position, showing the organization and the regional stress state is fluctuation. The grain of the HAZ grew up during thermal cycling, at the same time, the HAZ go through tissue expansion and contraction process, remaining larger residual stress[5]. The fusion line of the welding thermal cycle process of melting, solidification process experience part of the organization, the organization condition and the residual stress nonuniformity are large. The crystal structure of the fusion line is not uniform and Stress concentration in this zone. So the HAZ and adjacent to the fusion line easily become the weak link of the weld joint[6].Figure 7. Curves of crack growth rateFig. 7 shows the test of crack growth rate curves of the base metal, the WZ and HAZ. From Fig. 7, the slope of fatigue crack growth rate of HAZ is relatively high. When the stress intensity factor range Delta K reaches a certain value, the crack growth rate (da/dn) of HAZ is bigger than which of the WZ and base metal, becomes to the most dangerous area. Therefore, the following calculation focuses on the HAZ of crack initiation and propagation characteristics.The relationship between crack growth rate of fatigue crack propagation stage and the stress intensity factor range could represent by Paris law:da/dn=CΔK m(2) Combined with the crack growth rate curve of Fig. 7, the parameter C in the Paris law is 4.599×10-13, and m is 3.85183.Based on the BS7910:ΔK=YΔσ(πa) 1/2(3) In which, Y is the shape factor, can be calculated according to the BS7910. Delta sigma is the stress amplitude, using the peak value stress. Assuming that the crack length is from 0.1 extended to 0.5 mm, and then the Eq. 2 only has a unknown value, the life of fatigue crack growth of delta n. To obtain the fatigue crack propagation life, combined with the Eq. 1, the crack initiation life could be calculated.Figure 8. Curves of the crack propagation lifeThe crack initiation life and crack propagation life are shown in Fig. 8.As Fig. 8 shows, the fatigue crack initiation life accounted for most of the total fatigue life, and crack initiation life is negligible. Therefore, as the fatigue crack initiation, it may be quickly extended until fracture. Fatigue crack that once found, it should be immediately stoped using the welded conduit.4. ConclusionIn view of stainless steel 1Cr18Ni9Ti conduit, the crack growth rate test and fatigue life test were taken off. The characteristics of fatigue crack and fracture morphology were investigated. The conclusions are as follows:(1) The fatigue life of weld joints is able to meet the use requirements, the fatigue crack source in the surface of the conduit position, and the fault location is the weld toe. The weld toe position concentrated stress reduces the fatigue life of welded joints.(2) The stress state of HAZ and fusion line are not uniform, and the slope the crack growth rate curve is relatively bigger, which is considered to be the dangerous position of weld joint.(3) The HAZ of fatigue crack initiation life is accounted for most of the total fatigue life. As the fatigue crack initiation, it quickly expanded until rupture. Therefore, the results showed that the fatigue crack after welding conduit once be seen, it should immediately stop using.References[1] Y. Liu, H. Zhang, Z. zhao, Study on Damage tolerance of Stainless Steel Conduit in Aircraft.Aero Manu Tech, 13 (2012) 94–97.[2] P. J. Haagensen, Fatigue improvement techniques advantages and limitations. Weld World,47(2003) 43–63.[3] X. Gang, Effect of compressive surface stress on the fatigue of 10Ni5CrMoV steel. Devel AppMate,2( 2009) 1–3.[4] X. H. Cheng, J. W. Fisher, H. J. 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