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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.。

黄芩素对人乳腺癌细胞系MDA-MB-231_侵袭、迁移、上皮间充质转化的调控作用及其机制

黄芩素对人乳腺癌细胞系MDA -MB -231侵袭、迁移、上皮间充质转化的调控作用及其机制陈林1，2，梁秋果1，2，吉杨丹1，2，王恒1，21 黔南民族医学高等专科学校基础医学部，贵州都匀558013；2 黔南州天然产物与组分功效重点实验室摘要：目的　观察黄岑素对人乳腺癌细胞系MDA -MB -231的侵袭、迁移及上皮间充质转化（EMT ）的调控作用，探讨其可能作用机制。

方法　取对数生长期MDA -MB -231细胞分为一组、二组、三组及对照组，一组、二组、三组分别加入2.5、5、10 μmol /L 的黄岑素，对照组不做任何处理。

培养48 h 时采用划痕修复实验观察四组细胞迁移能力、采用Transwell 侵袭实验观察四组细胞侵袭能力，采用Western Blotting 法检测细胞EMT 标志物波形蛋白（vimentin ）及E -钙黏蛋白（E -cadherin ）、整合素αv 、β3、磷酸化黏着斑激酶（p -FAK ）、磷酸化磷脂酰肌醇3激酶（整合素p -PI3K ）。

结果　与对照组相比，黄岑素组细胞迁移率降低、侵袭细胞数少，细胞E -cadherin 相对表达量高，vimentin 、整合素αv 、整合素β3、p -FAK 、p -PI3K 蛋白相对表达量低，且呈剂量依赖性（P 均<0.05）。

结论　黄芩素抑制MDA -MB -231细胞的侵袭、迁移及EMT 。

黄岑素可能通过抑制整合素αv 、整合素β3表达，进一步抑制p -FAK 、p -PI3K 蛋白表达，抑制MDA -MB -231的侵袭、迁移及EMT 。

关键词：黄芩素；乳腺癌；细胞侵袭；细胞迁移；上皮间质转化；波形蛋白；E -钙黏蛋白；整合素αv 、整合素β3；黏着斑激酶；磷脂酰肌醇3激酶doi ：10.3969/j.issn.1002-266X.2024.06.003中图分类号：R965.1 文献标志码：A 文章编号：1002-266X （2024）06-0010-04Regulatory effects of baicalein on invasion ， migration ， and EMT in human breast cancer cell line MDA -MB -231CHEN Lin 1， LIANG Qiuguo ， JI Yangdan ， WANG Heng1 Department of Basic Medicine ， Qiannan Medical College for Nationalities ， Duyun 558013， ChinaAbstract ： Objective To investigate the regulatory effects of baicalein on the invasion ， migration ， and epithelial -mesenchymal transition （EMT ） of human breast cancer cell line MDA -MB -231 and to explore its potential mechanism of action. Methods MDA -MB -231 cells in the logarithmic growth phase were divided into the Group 1， Group 2， Group 3， and Control group. Baicalein was added to cells in the Group 1， Group 2， and Group 3 at concentrations of 2.5， 5， and 10 μmol /L ， respectively ， while cells in the Control group were not treated. After 48 h of incubation ， Scratch repair experi‐ment was used to observe cell migration capacity ， Transwell invasion experiment was performed to assess cell invasion ca‐pacity ， and Western blotting was utilized to detect the expression levels of EMT markers including vimentin and E -cad‐herin ， as well as integrin αv ， integrin β3， phosphorylated focal adhesion kinase （p -FAK ）， and phosphorylated phosphati‐dylinositol -3 kinase （p -PI3K ） proteins. Results Compared with the control group ， the cell migration rate decreased ， the number of invading cells decreased ， the relative expression level of E -cadherin was higher ， and the relative expression levels of vimentin ， integrin αv ， integrin β3， p -FAK ， and p -PI3K protein were lower in the baicalein group ， with a dose -de‐pendant manner （all P <0.05）. Conclusions Baicalein inhibits the invasion ， migration ， and EMT of MDA -MB -231cells. Baicalein may inhibit the expression of integrin αv ， integrin β3， and further inhibit the expression of p -FAK and p -PI3K proteins ， thereby inhibiting the invasion ， migration ， and EMT of MDA -MB -231 cells.Key words ： baicalein; breast carcinoma; cell invasion; cell migration; epithelial -mesenchymal transition; vimentin;基金项目：黔南民族医学高等专科学校校基金（qnyz202013，qnyz202206）；黔南民族医学高等专科学教育教学科研基金项目（qnyzjx202205）；黔南民族医学高等专科学校大学生科技创新项目（qnyz202201）。

超声引导下微波消融联合贝伐珠单抗治疗晚期结肠癌伴肝转移的临床价值

·临床研究·超声引导下微波消融联合贝伐珠单抗治疗晚期结肠癌伴肝转移的临床价值韩小军袁理郭道宁摘要目的探讨超声引导下微波消融联合贝伐珠单抗治疗晚期结肠癌伴肝转移的临床应用价值。

方法选取在我院就诊的102例晚期结肠癌伴肝转移患者，按随机数字表法分为观察组和对照组各51例，对照组采用贝伐珠单抗联合常规化疗治疗，观察组在此基础上采用超声引导下微波消融治疗；比较两组患者治疗后疗效、免疫功能、不良反应及预后情况。

结果治疗后，观察组客观缓解率（ORR）、疾病控制率（DCR）均高于对照组（均P<0.05）；两组CD3+、CD4+、CD8+均较治疗前下降，且观察组CD3+、CD4+、CD4+/CD8+均高于对照组，CD8+低于对照组，差异均有统计学意义（均P<0.05）。

治疗后，两组胃肠道反应、食欲减退、疲劳乏力等不良反应比较差异均无统计学意义；观察组累积无复发生存率及累积总生存率分别为78.77%、57.45%，均高于对照组（49.32%、34.23%），差异均有统计学意义（χ2=10.086、4.536，P=0.001、0.033）。

结论超声引导下微波消融联合贝伐珠单抗能提高晚期结肠癌伴肝转移患者的治疗效果，缓解免疫功能抑制，改善生存状况，具有较好的临床应用价值。

关键词超声引导；微波消融；结肠癌，晚期；肝转移；贝伐珠单抗［中图法分类号］R445.1［文献标识码］AClinical value of ultrasound-guided microwave ablation combined withbevacizumab in the treatment of advanced colonadenocarcinoma with liver metastasisHAN Xiaojun，YUAN Li，GUO DaoningDepartment of Ultrasound Medicine，Mianyang Hospital Affiliated to School of Medicine，University of Electronic Science andTechnology of China，Sichuan621000，ChinaABSTRACT Objective To explore the application clinical value of ultrasound-guided microwave ablation combined with bevacizumab in the treatment of advanced colon adenocarcinoma（COAD）with liver metastasis.Methods A total of102 patients with advanced COAD with liver metastasis treated in our hospital were selected，and divided into the observation group and the control group by random number table method，with51cases in each group.The control group was treated with bevacizumab combined with conventional chemotherapy.On this basis，the observation group was treated with ultrasound-guided microwave thermal ablation.The curative effect，immune function，adverse reactions and prognosis after treatment of the two groups were compared.Results After treatment，the objective remission rate（ORR）and disease control rate（DCR）in the observation group were higher than those in the control group（both P<0.05）.After treatment，the CD3+，CD4+and CD4/CD8+in the observation group were higher than those in the control group，and CD8+was lower than that in the control group，the differences were statistically significant（all P<0.05）.After treatment，there were no statistically significant difference in the incidence rates of adverse reactions such as gastrointestinal reactions，loss of appetite and fatigue between the two groups.The cumulative recurrence-free survival rate and cumulative overall survival rate in observation group were78.77%and57.45% respectively，which were significantly higher than those in control group（49.32%and34.23%），the differences were statistically significant（χ2=10.086，4.536，P=0.001，0.033）.Conclusion Ultrasound-guided microwave ablation combined with作者单位：621000四川省绵阳市，电子科技大学医学院附属绵阳医院绵阳市中心医院超声医学科（韩小军、郭道宁），肿瘤科（袁理）通讯作者：郭道宁，Email：******************结肠癌是常见的消化道肿瘤，近年来其发病率和死亡率均逐渐升高。

FUNDC1调控肺癌A549细胞及其干细胞的生长、自噬、凋亡和细胞干性的研究

本试验自中科院上海细胞研究所购入肺癌细胞系Ａ５４９、ＨＣＣ８２７、ＮＣＩ－Ｈ１６５０、ＮＣＩ－１９７５、ＰＣ９以及正常人肺上皮细胞ＢＥＡＳ－２Ｂ。细胞于５％ＣＯ２、３７℃恒温条件下接种于含有１００Ｕ／ｍＬ盘尼西林、１％链霉素和１０％胎牛血清的ＤＭＥＭ培养基中进行培养。
Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ实验被用来检测目标蛋白的表达水平。首先，将收集到的细胞在低温条件下使用含１％蛋白酶抑制
剂的预冷细胞裂解缓冲液进行裂解，４℃ １１４５１×ｇ离心３０ｍｉｎ，使用ＢＣＡ试剂盒（翊圣生物，上海）测定上清液中的蛋白含量。ＳＤＳ－ＰＡＧＥ（１０％）被用来分离等量的蛋白样本，随后进行转膜，并使用５％的脱脂牛奶于室温下封闭两小时。经过三次的ＴＢＳ缓冲液清洗后，一抗（均为兔抗多克隆抗体）过夜孵育蛋白膜（ａｎｔｉ－ＦＵＮＤＣ１、ａｎｔｉ－Ｂａｘ、ａｎｔｉ－Ｂｃｌ－２、ａｎｔｉ－ｃａｓｐａｓｅ３、ａｎｔｉ－ｐ６２、ａｎｔｉ－Ｂｅｃｌｉｎ－１、ａｎｔｉ－ＬＣ３－Ⅱ、ａｎｔｉ－ＣＤ１３３、ａｎｔｉ－Ｓｏｘ２、ａｎｔｉ－Ｏｃｔ４、ａｎｔｉ－β－ａｃｔｉｎ，稀释度１∶１０００，４℃），ＨＲＰ标记的山羊抗兔ＩｇＧ二抗孵育一小时（稀释度１∶２０００，３７℃）。所有抗体均购自Ａｂｃａｍ（上海）公司。添加ＥＣＬ试剂曝光显影蛋白条带１ｍｉｎ。β－ａｃｔｉｎ为本实验的内参蛋白。１．６ＣＣＫ－８细胞增殖实验
【收稿日期】２０２０－０９－２９【修回日期】２０２０－１１－２２【基金项目】陕西省西安市科技攻关项目［编号：２０１７１１３ＳＦ／ＹＸ００７（１）］【作者单位】西安市中心医院呼吸与危重症医学科，陕西西安
７１０００３【作者简介】董玉（１９７３－），女，贵州贵阳人，主任医师，研究方向：

丹参治疗肝癌分子机制的网络药理学探讨

丹参治疗肝癌分子机制的网络药理学探讨An network pharmacological study on molecular mechanism ofsalvia miltiorrhiza on liver cancer徐倩1曾柏荣2*（1.湖南中医药大学，湖南　长沙，410208；2.湖南中医药大学第一附属医院，湖南　长沙，410007）中图分类号：R285文献标识码：A文章编号：1674-7860（2020）31-0001-07证型：IAD【摘要】目的：基于网络药理学方法预测丹参治疗肝癌的可能作用靶点及机制。

方法：通过中药系统药理学数据库和分析平台（Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform，TCMSP）收集中药丹参的活性成分及其对应的靶基因，运用GeneCards数据库、DrugBank数据库获得肝癌相关靶基因，匹配共同基因作为丹参治疗肝癌的潜在靶标。

应用Cytoscape软件完成“中药-活性成分-作用靶点-疾病”网络，通过String数据库完成蛋白相互作用（Protein-protein interaction，PPI）网络，最后基于Metascape数据库进行基因本体论（Gene Ontology，GO）及京都基因和基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析。

结果：获得丹参酮ⅡA、隐丹参酮等59个丹参治疗肝癌的活性成分，作用于77个潜在靶标，预测可能与磷脂酰肌醇3-激酶/蛋白激酶B（Phosphatidylinositol 3-Kinase/Protein KinaseuB，PI3K-AKT）、乙型肝炎、人类T细胞白血病病毒1型（Human T Cell Leukemia Virus Type 1，HTLV-1）感染、癌症中的miRNAs 等信号通路有关。

Calcipotriol_SDS_MedChemExpress

Inhibitors, Agonists, Screening LibrariesSafety Data Sheet Revision Date:Sep.-13-2017Print Date:Sep.-13-20171. PRODUCT AND COMPANY IDENTIFICATION1.1 Product identifierProduct name :CalcipotriolCatalog No. :HY-10001CAS No. :112965-21-61.2 Relevant identified uses of the substance or mixture and uses advised againstIdentified uses :Laboratory chemicals, manufacture of substances.1.3 Details of the supplier of the safety data sheetCompany:MedChemExpress USATel:609-228-6898Fax:609-228-5909E-mail:sales@1.4 Emergency telephone numberEmergency Phone #:609-228-68982. HAZARDS IDENTIFICATION2.1 Classification of the substance or mixtureNot a hazardous substance or mixture.2.2 GHS Label elements, including precautionary statementsNot a hazardous substance or mixture.2.3 Other hazardsNone.3. COMPOSITION/INFORMATION ON INGREDIENTS3.1 SubstancesSynonyms:MC 903; CalcipotrieneFormula:C27H40O3Molecular Weight:412.60CAS No. :112965-21-64. FIRST AID MEASURES4.1 Description of first aid measuresEye contactRemove any contact lenses, locate eye-wash station, and flush eyes immediately with large amounts of water. Separate eyelids with fingers to ensure adequate flushing. Promptly call a physician.Skin contactRinse skin thoroughly with large amounts of water. Remove contaminated clothing and shoes and call a physician.InhalationImmediately relocate self or casualty to fresh air. If breathing is difficult, give cardiopulmonary resuscitation (CPR). Avoid mouth-to-mouth resuscitation.IngestionWash out mouth with water; Do NOT induce vomiting; call a physician.4.2 Most important symptoms and effects, both acute and delayedThe most important known symptoms and effects are described in the labelling (see section 2.2).4.3 Indication of any immediate medical attention and special treatment neededTreat symptomatically.5. FIRE FIGHTING MEASURES5.1 Extinguishing mediaSuitable extinguishing mediaUse water spray, dry chemical, foam, and carbon dioxide fire extinguisher.5.2 Special hazards arising from the substance or mixtureDuring combustion, may emit irritant fumes.5.3 Advice for firefightersWear self-contained breathing apparatus and protective clothing.6. ACCIDENTAL RELEASE MEASURES6.1 Personal precautions, protective equipment and emergency proceduresUse full personal protective equipment. Avoid breathing vapors, mist, dust or gas. Ensure adequate ventilation. Evacuate personnel to safe areas.Refer to protective measures listed in sections 8.6.2 Environmental precautionsTry to prevent further leakage or spillage. Keep the product away from drains or water courses.6.3 Methods and materials for containment and cleaning upAbsorb solutions with finely-powdered liquid-binding material (diatomite, universal binders); Decontaminate surfaces and equipment by scrubbing with alcohol; Dispose of contaminated material according to Section 13.7. HANDLING AND STORAGE7.1 Precautions for safe handlingAvoid inhalation, contact with eyes and skin. Avoid dust and aerosol formation. Use only in areas with appropriate exhaust ventilation.7.2 Conditions for safe storage, including any incompatibilitiesKeep container tightly sealed in cool, well-ventilated area. Keep away from direct sunlight and sources of ignition.Recommended storage temperature: 4°C, protect from light, stored under nitrogenShipping at room temperature if less than 2 weeks.7.3 Specific end use(s)No data available.8. EXPOSURE CONTROLS/PERSONAL PROTECTION8.1 Control parametersComponents with workplace control parametersThis product contains no substances with occupational exposure limit values.8.2 Exposure controlsEngineering controlsEnsure adequate ventilation. Provide accessible safety shower and eye wash station.Personal protective equipmentEye protection Safety goggles with side-shields.Hand protection Protective gloves.Skin and body protection Impervious clothing.Respiratory protection Suitable respirator.Environmental exposure controls Keep the product away from drains, water courses or the soil. Cleanspillages in a safe way as soon as possible.9. PHYSICAL AND CHEMICAL PROPERTIES9.1 Information on basic physical and chemical propertiesAppearance White to off-white (Solid)Odor No data availableOdor threshold No data availablepH No data availableMelting/freezing point No data availableBoiling point/range No data availableFlash point No data availableEvaporation rate No data availableFlammability (solid, gas)No data availableUpper/lower flammability or explosive limits No data availableVapor pressure No data availableVapor density No data availableRelative density No data availableWater Solubility No data availablePartition coefficient No data availableAuto-ignition temperature No data availableDecomposition temperature No data availableViscosity No data availableExplosive properties No data availableOxidizing properties No data available9.2 Other safety informationNo data available.10. STABILITY AND REACTIVITY10.1 ReactivityNo data available.10.2 Chemical stabilityStable under recommended storage conditions.10.3 Possibility of hazardous reactionsNo data available.10.4 Conditions to avoidNo data available.10.5 Incompatible materialsStrong acids/alkalis, strong oxidising/reducing agents.10.6 Hazardous decomposition productsUnder fire conditions, may decompose and emit toxic fumes.Other decomposition products - no data available.11.TOXICOLOGICAL INFORMATION11.1 Information on toxicological effectsAcute toxicityClassified based on available data. For more details, see section 2Skin corrosion/irritationClassified based on available data. For more details, see section 2Serious eye damage/irritationClassified based on available data. For more details, see section 2Respiratory or skin sensitizationClassified based on available data. For more details, see section 2Germ cell mutagenicityClassified based on available data. For more details, see section 2CarcinogenicityIARC: No component of this product present at a level equal to or greater than 0.1% is identified as probable, possible or confirmed human carcinogen by IARC.ACGIH: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by ACGIH.NTP: No component of this product present at a level equal to or greater than 0.1% is identified as a anticipated or confirmed carcinogen by NTP.OSHA: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by OSHA.Reproductive toxicityClassified based on available data. For more details, see section 2Specific target organ toxicity - single exposureClassified based on available data. For more details, see section 2Specific target organ toxicity - repeated exposureClassified based on available data. For more details, see section 2Aspiration hazardClassified based on available data. For more details, see section 212. ECOLOGICAL INFORMATION12.1 ToxicityNo data available.12.2 Persistence and degradabilityNo data available.12.3 Bioaccumlative potentialNo data available.12.4 Mobility in soilNo data available.12.5 Results of PBT and vPvB assessmentPBT/vPvB assessment unavailable as chemical safety assessment not required or not conducted.12.6 Other adverse effectsNo data available.13. DISPOSAL CONSIDERATIONS13.1 Waste treatment methodsProductDispose substance in accordance with prevailing country, federal, state and local regulations.Contaminated packagingConduct recycling or disposal in accordance with prevailing country, federal, state and local regulations.14. TRANSPORT INFORMATIONDOT (US)This substance is considered to be non-hazardous for transport.IMDGThis substance is considered to be non-hazardous for transport.IATAThis substance is considered to be non-hazardous for transport.15. REGULATORY INFORMATIONSARA 302 Components:No chemicals in this material are subject to the reporting requirements of SARA Title III, Section 302.SARA 313 Components:This material does not contain any chemical components with known CAS numbers that exceed the threshold (De Minimis) reporting levels established by SARA Title III, Section 313.SARA 311/312 Hazards:No SARA Hazards.Massachusetts Right To Know Components:No components are subject to the Massachusetts Right to Know Act.Pennsylvania Right To Know Components:No components are subject to the Pennsylvania Right to Know Act.New Jersey Right To Know Components:No components are subject to the New Jersey Right to Know Act.California Prop. 65 Components:This product does not contain any chemicals known to State of California to cause cancer, birth defects, or anyother reproductive harm.16. OTHER INFORMATIONCopyright 2017 MedChemExpress. The above information is correct to the best of our present knowledge but does not purport to be all inclusive and should be used only as a guide. The product is for research use only and for experienced personnel. It must only be handled by suitably qualified experienced scientists in appropriately equipped and authorized facilities. The burden of safe use of this material rests entirely with the user. MedChemExpress disclaims all liability for any damage resulting from handling or from contact with this product.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。

益生菌对阿尔茨海默病作用的研究进展

益生菌对阿尔茨海默病作用的研究进展发布时间：2021-12-14T06:08:15.523Z 来源：《中国结合医学杂志》2021年12期作者：宋鑫萍1,2，李盛钰2，金清1[导读] 阿尔茨海默病已成为威胁全球老年人生命健康的主要疾病之一，患者数量逐年攀升，其护理的经济成本高，给全球经济造成重大挑战。

近年来研究显示，益生菌在适量使用时作为有益于宿主健康的微生物，在防治阿尔茨海默病方面具有积极影响，其作用机制可能通过调节肠道菌群，影响神经免疫系统，调控神经活性物质以及代谢产物，通过肠-脑轴影响该病发生和发展。

宋鑫萍1,2，李盛钰2，金清11.延边大学农学院，吉林延吉 1330022.吉林省农业科学院农产品加工研究所，吉林长春 130033摘要：阿尔茨海默病已成为威胁全球老年人生命健康的主要疾病之一，患者数量逐年攀升，其护理的经济成本高，给全球经济造成重大挑战。

本文综述了近几年来国内外益生菌对阿尔茨海默病的作用进展，以及其预防和治疗阿尔茨海默病的潜在作用机制。

关键词：益生菌；阿尔茨海默病；肠道菌群；机制Recent Progress in Research on Probiotics Effect on Alzheimer’s DiseaseSONG Xinping1,2，LI Shengyu2，JI Qing1*(1.College of Agricultural, Yanbian University, Yanji 133002，China)(2.Institute of Agro-food Technology, Jilin Academy of Agricultural Sciences, Chanchun 130033, China)Abstract：Alzheimer’s disease has become one of the major diseases threatening the life and health of the global elderly. The number of patients is increasing year by year, and the economic cost of nursing is high, which poses a major challenge to the global economy. In recent years, studies have shown that probiotics, as microorganisms beneficial to the health of the host, have a positive impact on the prevention and treatment of Alzheimer’s disease. Its mechanism may be through regulating intestinal flora, affecting the nervous immune system, regulating the neuroactive substances and metabolites, and affecting the occurrence and development of the disease through thegut- brain axis. This paper reviews the progress of probiotics on Alzheimer’s disease at home and abroad in recent years, as well as its potential mechanism of prevention and treatment.Key words：probiotics; Alzheimer’s disease; gut microbiota; mechanism阿尔茨海默病（Alzheimer’s disease, AD），系中枢神经系统退行性疾病，属于老年期痴呆常见类型，临床特征主要包括：记忆力减退、认知功能障碍、行为改变、焦虑和抑郁等。

219316022_基于1H_NMR代谢组学技术的黄刺玫果实抗衰老作用研究

牛丹，陈磊，任婧，等. 基于1H NMR 代谢组学技术的黄刺玫果实抗衰老作用研究[J]. 食品工业科技，2023，44（12）：10−17. doi:10.13386/j.issn1002-0306.2022080287NIU Dan, CHEN Lei, REN Jing, et al. Research on the Anti-aging Effect of Rosa xanthina Lindl Fruits Based on 1H NMR Metabolomics[J]. Science and Technology of Food Industry, 2023, 44(12): 10−17. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2022080287· 未来食品 ·基于1H NMR 代谢组学技术的黄刺玫果实抗衰老作用研究牛　丹1，陈　磊2，任　婧1，王进东1,*（1.山西省药品审评中心（山西省医药与生命科学研究院），山西太原 030006；2.山西省药品检查中心（山西省疫苗检查中心），山西太原 030031）摘　要：目的：采用1H NMR 代谢组学方法，探讨黄刺玫果实对D-半乳糖致衰老模型大鼠的干预作用。

方法：皮下注射D-半乳糖（400 mg/kg ）致大鼠亚急性衰老，考察黄刺玫果实提取物（1.6、3.2、6.4 g/kg ）的抗衰老作用。

造模30 d 后采集大鼠血清，检测血清生化指标SOD （superoxide dismutase ）、LPO （lipid peroxide ）及Hyp （hydroxyproline ）水平，并进行1H NMR 代谢组学检测，结合多元统计分析研究黄刺玫果实提取物高、中、低剂量的抗衰老作用。

结果：与模型组相比，黄刺玫果实提取物可显著升高血清SOD （P <0.01）、Hyp （P <0.01）水平，降低LPO （P <0.05，P <0.01）水平。

辛伐他汀通过抑制类异戊二烯的合成降低TDP-25细胞和hSOD1G93A细胞的活力

脑与神经疾病杂志2021年第29卷第3期133•论著.辛伐他汀通过抑制类异戊二烯的合成降低TDP-25细胞和hSODlG93A细胞的活力齐伟静刘亚玲李睿杨帅李帅耿丽颖张欣钱倩【摘要】目的观察辛伐他汀对肌萎缩侧索硬化(ALS)的两种细胞模型：TDP-25细胞和hSODlG93A细胞活力的影响c方法辛伐他汀、辛伐他汀联合甲轻戊酸途径的下游产物以及下游抑制剂分别干预TDP-25细胞和hSODlG93A细胞，应用CCK-8法和LDH法检测细胞活力的变化。

结果辛伐他汀降低了TDP25细胞和hSODlG93A细胞的活力，呈剂量依赖性和时间依赖性。

辛伐他汀导致的细胞活力下降可以通过补充甲疑戊酸，FPP或GGPP而完全缓解，但补充胆固醇不能。

另外，FTI-277可以明显降低TDP-25细胞的活力，GGTI-286可以同时降低TDP-25细胞和hSODlG93A细胞的活力，而胆固醇抑制剂AY9944对两种细胞的活力均无明显影响。

结论辛伐他汀导致TDP-25细胞和hSODlG93A细胞的活力下降，这种细胞毒性作用可能是由于抑制了类异戊二烯的合成。

【关键词】辛伐他汀；肌萎缩侧索硬化；类异戊二烯；TDP25细胞；hSODlG93A细胞中图分类号：R744.8文献标识码：A文章编号：1006-351X(2021)03-0133-05Simvastatin decreased cell viability of TDP-25cells and hSODlG93A cells by inhibiting isoprenoid synthesisQi Weijing,Liu Yaling,Li Rui,Yang Shuai,Li Shuai,Geng Liying,Zhang Xin,Qian QianDepartment of N eurology,the Baoding No.l Central Hospital,Hebei071000,ChinaCorresponding author:Liu Yaling,Email:lyldoctor@[Abstract]Objective To observe the effect of simvastatin on cell viability of amyotrophic lateral sclerosis(ALS)cell models:TDP-25cells and hSODlG93A cells.Methods Simvastatin combined with mevalonate pathway products,and mevalonate pathway downstream inhibitors were used to incubate TDP-25cells and hSODlG93A K-8and LDH were used to detect changes in cell viability.Results Simvastatin decreased cell viability of TDP-25cells andhSODlG93A cells in a dose-and time-dependent manner.The addition of mevalonate,FPP or GGPP could improve thecell damage caused by simvastatin,whereas cholesterol could not reverse the toxic effect of simvastatin.FTI-277couldinduce a significant decrease in cell viability of TDP-25cells,GGTI-286significantly decreased cell viability of TDP-25cells and hSODlG93A cells,while AY9944had no significant effect on cell viability of TDP-25cells or hSODlG93Acells.Conclusions Simvastatin decreased cell viability of TDP-25cells and hSODlG93A cells,and these cytotoxic effectswere related to the inhibition of isoprenoid synthesis.[Key words]Simvastatin;Amyotrophic lateral sclerosis；Isoprenoid;TDP-25cell:hSODlG93A cell肌萎缩侧索硬化(amyotrophic lateral sclerosis, ALS)是一种致命的神经变性疾病，通过选择性损伤脊髓、脑干和大脑皮质中的运动神经元Hi,导致患者出现进行性的肌无力、肌萎缩等，多在发病基金项目：国家自然科学基金(81871001),河北省卫生健康委科研基金项目(20200265)作者单位:071000河北，保定市第一中心医院神经内科(齐伟静、杨帅、耿丽颖、张欣、钱倩)；河北医科大学第二医院神经内科，河北省神经病学重点实验室(刘亚玲、李睿、李帅)通信作者：刘亚玲，Email：*****************2~5年内因呼吸衰竭死亡。

Keap1

非小细胞肺癌(non-small cell lung cancer，NSCLC)发病率占据肺癌的75%~80%。

肿瘤细胞进展快且易扩散转移，临床常采用手术、放化疗等进行治疗，但5年生存率低于60%[1-2]。

氧化应激是由活性氧(ROS)生成量增加所致，ROS积累可诱导肺癌细胞凋亡，清除ROS 可阻止癌细胞凋亡，即肺癌细胞存活依赖于癌细胞自身抗氧化能力[3]。

Kelch样环氧氯丙烷相关蛋白-1 (kelch-like epichlorohydrin-associated protein-1，Keap1)/核因子E2相关因子2(nuclear factor E2related factor 2，Nrf2)信号通路在癌症中发挥重要调控作用，氧化应激可激活Keap1，促使Keap1-Nrf2复合物裂解，Nrf2转移至细胞核内，可激活下游靶基因表达，参与肺癌发生发展过程[4]。

Nrf2可维持氧化还原稳态，ROS侵袭细胞时，Nrf2可进入细胞核，结合抗氧化反应元件(ARE)转录编码各种抗氧化蛋白、代谢酶基因，抑制氧化应激反应[5-6]。

目前氧化应激、Keap1/Nrf2信号通路在NSCLC发生过程中的机制尚未明确。

基于此，本研究尝试分析Keap1/Nrf2信号通路与临床病理参数、氧化应激指标的相关性，探讨其在NSCLC氧化应激机制中的作用，为临床研制新药提供参考依据。

1资料与方法1.1一般资料选取2017年4月至2020年4月郑州市第三人民医院收治的100例NSCLC患者为研究对象。

纳入标准：符合NSCLC诊断标准[7]；术前未接受放化疗、免疫治疗者；预计生存期≥6个月；符合手术适应证、禁忌证；Karnofsky功能状态评分≥70分；签署知情同意书。

排除标准：合并凝血功能障碍、肝肾功能障碍、其他恶性肿瘤者；伴有急/慢性感染者；伴有精神疾病者；既往腹部相关外科手术史者。

所有患者均行肺癌根治性切除术，术中收集癌组织、癌旁组织(距离癌组织5cm范围内正常组织)，其中男性63例，女性37例；年龄46~67岁，平均(56.32±3.16)岁；体质量指数(BMI)17~30kg/m2，平均(23.16±2.03)kg/m2；病理类型：鳞癌58例、腺癌42例；病理分级[8]：Ⅰ~Ⅱ级51例、Ⅲ级49例；T分期[9]：T1~T253例、T3~T447例；N分期：N055例、N1~N245例。

新型镇痛药ω-芋螺毒素的合成等效物Ziconotide

新型镇痛药ω-芋螺毒素的合成等效物ZiconotideChineseJournalofNewDrugs2005,V o1.14No4[2][3][4][5][6][7][8][9][参考文献]RashidM,ManivetP,NishioH,etalIdentificationofthebind—ingsitesandselectivityofsarpogrelate,anovel5一HT2antagonist,tohuman5-HT2A,5-HT2Band5-HT2freceptorsubtypesby molecularmodeling[JjlLSci,2003,73(2):193207.GrayJA,RothBLParadoxicaltraffickingandregulationof5一HT(2A)receptorsbyagonistsandantagonists[J]BrainResBull,2001,56(5):441451QiR,OzakiS,SatohK,etalQuantitativemeasurementofvariOUS5一HTreceptorantagonistsonplateletactivationinducedbyserotonin[J].ThrombRes,1996,81(1):43—54.NakawaK,KariyazonoH,MasudaH.eta1.Effectofsarpogre—latehydrochlorideonadenosinediphosphateorcollagen—induced plateletresponsesinarteriosclerosisobliterans[J].B,)(g"Fibrinolysis,2002,12(5):391397KandabashiT,ShimokawaH,MukaiY.etalInvolvementofrho—kinaseinagonists—inducedcontractionsofarterioscler0tichu marlarteries【Jj.ArteriosclerThrombV ascBiol,2002,22(2): 243248.MikawaK,AkamatsuH,NishinaK.etalTheeffectsofsar—pogrelateonsuperoxideproductionbyhumanneutrophils[J]. RegAnesthPainMed,2000,25(2):181—186.Y amashitaT,KitamoriK,HashimotoM.eta1.Conjunctiveef—fectsofthe5HT2receptorantagonist,sarpogrelate,oHthrombol—ysiswithmodifiedtissueplasminogenactivatorindifferentlaser—inducedthrombosismodels[J]Haemostasis,2000,30(6): 321332.SatomuraK,TakaseB,HamabeA,eta1.Sarpogrelate,aspecific5-HTreceptorantagonist,imporvesthecoronarymicrocirculation incoronaryarterydisease[J].ClinCardiol,2002,25(1):2832.SetoguchiY,OhnukiT,RashidM,eta1.Effectofchronicad—ministrationofsarpogrelateonsystolicbloodpressureofsponta一[10][12][13][14][15][16][17]中国新药杂志2005年第14卷第4期neouslyhypertensiverats:comparisonwithquinapril[J]Phar—macology,2002,64(2):71—75IgarashiM,OkudaT,KogaM,etalChangesinplasmasero—toninconcentrationandaccelerationplethysmogramsinpatientswithRaynaud'sphenomenonafterlong—termtreatmentwitha5一HTreceptorantagonist[Jj.JDermatol,2000,27(10):643650.IsrailovaM,SuzukiF,TanakaT,eta1.Bindingandfunctional affinityofsarpogrelate,itsmetabolitem一1andketanserinforhu—manrecombinantalpha一1adrenoceptorsubtypes[J].Pharnlacol—ogy,2002,65(2):69—73BrasilD,TemsahR,KumarK,etalBlockadeof5一HT(2A)re—ceptorsbysarpogrelateprotectstheheartagainstmycardioalin farctioninrats[J].JCardiovascPharmacolThPr,2002,7(1):5359FujitaM,MizunoK,MamiH,etalSarpogrelatetreatmentre—ducesrestenosisaftercoronarystenting[J]AmHeartJ,2003,145(2):el6WatanabeT,PakalaR,KatagiriT,etalMonocytechemotactic protein1amplifiesserotonininducedvascularsmoothmusclecell proliferation[J]JV ascRes,2001,38(4):341—349OriguchiN,ShigematsuH,MutoTAnplag,aselective5HTre—ceptorantagonist,reducesstenosisinducedbyballoninjuryinthe hypercholesterolaemicrabbit[JjlntAngiol,1997,16(3): 204—209.HayashiT,SumiD,MutoT,etalSarpogrelateHCI,aselective5HTreceptorantagonist.retardstheprogressionofarterioscle—rosisthroughanovelmechasm[J]Arteriosclerosis,2003,168 (1):2331DoggrellSA.TheroleOf5一HTonthecardiovascularandrenal systemsandtheclinicalpotentialof5HTmodulation[J:.Expert OpinlnvestigDrugs,2003,12(5):805823(接受日期:200410—30)新型镇痛药∞.芋螺毒素的合成等效物Ziconotide陈时宏(厦门北大之路生物工程有限公司厦门生物工程技术研究中心,厦门361009) [摘要]m一芋螺毒素的合成等效物Ziconotide作为一种新型非吗啡类镇痛剂,是首个应用于临床的具有神经元特异性的N一型电压敏感型性钙通道阻滞剂.大量临床前和临床资料证明,通过鞘内注射Ziconotide治疗严重慢性疼痛,可缓解其他治疗手段包括鞘内注射吗啡无效的疼痛;且长时间使用该药物不会产生耐受性和成瘾性.[关键词]Ziconotide;鞘内注射;N一型电压敏感性钙通道阻滞剂;镇痛作用[中图分类号]R971.2;R914.5[文献标识码]A[文章编号]1003—3734(2005)040490—06Ziconotide,anovelantin0ciceptiVeagentCHENShi—hong(XialllenResearchCenterofBiotechnology,XiamenPKUBioPark,Xiamen361009.China )[Abstract]Ziconotideisanovelnon—opioidanalgesics,developedforthetreatmentofseverechronicpain.Ziconotideisthesyntheticequivalentoftheconesnailpeptide(D—conotoxinMVIIA,the--——490?--——ChineseJournalofNewDrugs2005,V o1.14No.4主旦新药苤查笙第鲞箜塑firstofaneurone—specificN—typevoltage—sensitivecalciumchannelblocker.Clinicalstudiesdemonstrat—edthatintratheralziconotidemaybeeffectiveinpatientswithpainthatisrefractorytoopioidth erapyorthosewithintolerableopioid—relatedadverseeffects.Therewasnoevidenceoftolerancetoziconotideorofaddictivebehaviorinanimalsandhuman.[Keywords]ziconotide;intrathecaladministration;N—typevoltage—sensitivecalciumchannelblocker;antinociceptiveeffectZiconotide是首个应用于临床的具有神经元特异性(neurone—specific)的N一型电压敏感型性钙通道(voltage—sensitivecalciumchannel,VSCC)阻滞剂¨.作为一种新型非吗啡类镇痛剂,该药物(商品名Prialt)已分别于2004年底和2005年初获得美国FDA和欧盟(EC)的上市许可_2-3.通过鞘内(in—tratheca1)输注本品用于治疗严重慢性疼痛,可缓解其他治疗手段包括鞘内注射吗啡无效的疼痛;且长时间使用该药物不会产生耐受性和成瘾性.1化学结构和性质本品是25个氨基酸组成的聚合阳离子肽(polycationicpeptide)_1,43,含有的碱性氨基酸包括4个赖氨酸和2个精氨酸;等电点pI是l1.2l5,在体液中以阳离子形式存在.本品的分子式是C02H.72N6O2,相对分子质量是2639.12,CAS化学物质登记号是107452—89—1,氨基酸序列如下:H—Cys—Lys..Gly..Lys..Gly..Ala..Lys..Cys..Ser..Arg..Leu..Met..Tyr..Asp..Cys??Cys??Thr??Gly??Ser??Cys??Arg??Ser??Gly—-Lys??Cys??Hecyclic(1.16),(8—20),(15—25)一tris(disulfide)一.本品分子中的6个半胱氨酸残基通过3对二硫键相连的结构对其药理活性起重要作用,.其三维空间结构已用二维核磁共振波谱测定出;通过构效分析已确定其与钙通道结合的药效团_l,'7l:2位的赖氨酸残基,10位和21位的精氨酸以及N一端是该分子与N一型VSCC相互作用的重要部位l5.2作用机制N一型VS(,C阻滞剂作为一类新型药物,可选择性地阻断负责疼痛信号传导的神经通道,从而发挥镇痛作用.按结构和电生理的角度,VSCCs至少包括5种类型:L,N,T,P和Q型.其中N一型VSCC是神经细胞所特有的,它可调节去极化引起的钙离子内流和控制一些钙离子依赖过程包括神经元兴奋性和神经递质的释放l7.本品是∞一芋螺毒素(∞一conotoxinMVⅡA)的合成等效物,该毒素多肽是从一种食鱼芋螺(Conus magus)分泌的毒素中分离出的.动物研究表明:本品与N一型VSCC有着高度的亲和力(Kn=10pmol? L)J,能特异性地结合并阻断N一型VSCC,阻断钙内流,该作用具有可逆性.鞘内注射本品所产生的镇痛作用是通过阻断神经递质从主要的伤害感受性传人神经(primarynociceptiveafferents)释放并阻止疼痛信号传播至脑.3临床前安全性评价毒理学研究表明,在高于临床人用剂量下,本品无器官特异性,无致突变和致畸作用l8.在28d的亚急性毒性实验中_4,犬和大鼠对鞘内持续恒速滴注28d的高剂量(分别是1500和300ng?kg～?h)具有很好的耐受性.鞘内长期滴注实验表明,本品不影响临床血液学和生化指标,也无指示靶器官毒性作用的组织病理学方面的发现.动物静脉,脑室内或鞘内途径给予高剂量本品后会出现剂量依赖性的摇晃行为(shakingbehav—ior),共济失调,身体失衡和活动力(1ocomotoractivi—ty)增加等,一般可维持整个滴注过程,随着治疗的继续,这些反应会减弱直至停止,滴注后完全恢复.以上作用是∞一芋螺毒素家族中的N.型VSCC阻滞剂的一个特征l4.清醒大鼠静脉注射本品后可使全身血压剂量依赖性降低_l,,这主要是由于拟交感神经递质释放的阻断以及肥大细胞释放组胺引起的.该化合物的抗交感作用部分是通过阻断神经节后拟去甲肾上腺素能神经末梢去极化引起的钙内流及抑制其后的去甲.肾上腺素的释放.虽然由于抗交感作用引起的全身血压下降的静脉给药的ED50很低(约12g? kg);但在啮齿类动物的急性,持续性和神经性疼痛模型中,该剂量还是比产生镇痛作用的鞘内途径所需剂量要高许多.例如在大鼠脊神经结扎引起的外周神经病模型中,使得触觉超敏反应的阈值提高50%的鞘内药物剂量是0.3g?kg;并且由于该药的缓慢再分布,鞘内高剂量注射本品不会引起低血压.当以高于产生最大镇痛作用2～3倍的剂量鞘内单次注射本品时,未发现全身血压的变化.一491—ChineseJournalofNewDrugs2005,V ol14No44临床前药效学研究4.1离体实验[]见表l.表1有关Ziconotide的离体实验模型和实验结果克隆细胞株表达成孔蛋白和大鼠一型钙通道的电压感应亚单位分化的人成神经细胞瘤IMR32细胞中的钙离子流大鼠脑突触体膜小鼠神经肌肉接头结合实验显示Ziconotide对.型VSCC作用的特异性低浓度FZiconotide不可逆阻断钙离于流:10m~ol?LI1阻断(4214)%;但在高浓度下呈不完全阻断,约80%l,Ziconotide能部分阻断去甲肾上腺素释放.但不影响氨基丁酸和谷氨酸的释放Ziconotide与N一型通道的结合呈阳性反应Ziconotide对非N一型突触前Ⅵ℃无影响4.2整体动物模型实验不同的急慢性疼痛的动物模型均证明了本品的镇痛作用,包括甲醛|l卜,热板[¨,12,H],爪压力[H,浸尾[H,神经性疼痛(结扎脊神经|4,或局部坐骨神经ll一),紫外灼伤|4一,慢性缩窄性神经损伤161和切割性术后疼痛l17_等实验模型.动物实验表明,本品不产生耐受性和成瘾性,且与吗啡之间不产生交叉耐受性,两药物合用呈协同作用,并且因为本品不会阻止吗啡耐受性的产生使得该协同作用随时问的增加而减弱|4,14j.5药动学研究5.1动物药动学实验在大鼠和猕猴的药动学实验中|l,24h持续恒速静脉滴注本品,血浆药物浓度用放射免疫分析法测定.大鼠的滴注速度是2mL?kg?h～,在0.417和1.67mg?kgI.h剂量下的Css分别是1.1和4.2mg?L～,停止滴注后18h,超过99%的药物从血浆中清除.猴子的滴注速度是lmL?kg?h,在0.467和1.04mg?kg.?h剂量下的Css分别是1.4和3.2mg?L.结果显示两种动物的药动学参数很相近,药动学参数无剂量和性别依赖性;开始滴注后的2～4h内,到达稳态血浓,数据符合二房室模型;稳态时的分布体积约为体重的40%,表明血管外药物分布于细胞外和细胞内的体液中;本品与血浆蛋白的结合较低(大鼠和猴分别是56%和73%),非特异性,非饱和性和低亲和力,因此不可能导致药物在血管内的滞留;消除曲线是双指数曲线,大鼠和猕猴快速消除相的t和t/2a分别是0.375和0.730h.绝大部分的AUC与药物分布中的快速消除相有关,约占AUC的97%. 大鼠和猴的明显终末半衰期分别是4.6l和6.48h. 大鼠在滴注开始lh内,高低剂量组分别约有93% 和l7%出现轻微震颤;猕猴在滴注开始0.5h内,所有高剂量组和50%低剂量组出现轻微震颤.在犬的药动学研究l6J中,鞘内单次注射本品后药物在脑脊液中快速分布,且其后快速分布至脊髓.--——492.--——中国新药杂志2005年第14卷第4期和其他组织.鞘内持续滴注本品后其在脑脊液的量比血浆中的高l000倍,表明本品主要分布在其靶作用点.实验中结合使用H一inulin显示,本品在脑脊液中的清除速度与脑脊液本身的总体流动(bulk flow)接近.鞘内途径给予的本品在血浆中消除迅速,因此导致相当少的血浆蛋白结合|8一.静脉途径的本品在大鼠脑组织中2～24h内降解,产生的中间代谢产物检测不到,并且迅速从脑脊液和循环系统清除.这种快速的清除率可能暗示本品在脑脊液中的分布和它在脑脊液中的代谢也是快速的|8一.5.2人体药动学试验健康男性志愿者静脉持续滴注本品的血浆药物浓度一时间数据表明,该药物符合线性消除的二房室开放性药动学模型.血浆稳态浓度与滴注速率成比例.消除半衰期为1.3h,T.约为5.3h|l,.在Ⅱ期开放性临床试验中|6,研究22例慢性神经性疼痛患者给予鞘内途径的本品48h内脑脊液中的药动学(PK),以及其在血浆和脑脊液中的浓度与安全性和有效性的关系(PK—PD关系).鞘内单次给予本品l,5,7.5和lOt,g?kg,结果显示脑脊液药物浓度曲线可能呈多房室特征;剂量标准化AUC..,C.,CL,Vd和tl/2等与剂量成比例,提示本品在脑脊液的分布不随剂量增加而出现饱和现象.本品在血浆和脑脊液中呈线性的清除过程ll,与其以1～lOt,g?kg?d静脉滴注时血浆中的特征一致|l.脑脊液CL的平均值和中间值分别是0.38和0.26mL?min..成人脑脊液形成或消除的速度是0.35～0.37mL?min,即本品在脑脊液中的清除速度与脑脊液本身的总体流动或称周转率接近l8.脑脊液的平均值和中间值分别是155和99mL,均与成人的脑脊液体积约(130±30)mL接近.脑脊液的AUC../13平均值是70.8ng?h? mL.?g.,tl/2是4.6h,C/13是15.0ng?mL.?g～.脑脊液与血浆中的药物浓度比是303～l165.PK—PD有相关性.6临床研究本品的临床研究表明,该药安全,有效,且具有良好的耐受性.6.1I期临床试验在I期临床试验中,采用双盲和安慰剂对照法等,主要考察本品包括静脉给药等的安全性和耐受性.试验中剂量的提高是以安全性和耐受性评价为基础的.40例健康男性志愿者有30例持续24h以上恒ChineseJournalofNewDrugs2005,V o1.14No.4速静脉滴注本品0.3～10tLg?kg?d一,其余的给予安慰剂[一.主要不良反应是由外周交感神经阻断引起的心血管作用,如直立性低血压和窦性心动过缓,其中最主要的是直立性低血压,在所给的剂量范围内与剂量相关,高剂量组全部产生与直立性低血压有关的眩晕.56例健康男性志愿者有42例持续24h静脉滴注本品0.01～1.0mg?kg?d一,其余的给予安慰剂[I'z.一.所有给药组均出现由外周交感神经阻断引起的直立性低血压,但仰卧位的收缩压和舒张压及心率的变化是中等程度.当剂量高达lmg?kgd时,该药安全且可耐受.人静脉滴注的最大耐受剂量大于lmg?kg?d～;人药动学预试验表明,剂量小于42tLg?kg?h时血药浓度为15ng?mL～[18i.96例健康男性志愿者中72例持续24h静脉滴注本品0.3～1.0mg?kg?d～,其余的给予安慰剂_4.试验中受试者对本品一般可很好地耐受,但会产生直立性低血压,表现为头昏眼花和昏厥.试验表明健康受试者在静息状态下的血压的降低是适度和可耐受的.为更全面了解本品对人体心血管系统的影响,对持续6h静脉滴注1.0mg?kg?d0的高血压和正常血压的志愿者进行侵入性血液动力学监控_4一.结果表明,静脉注射本品引起的血压降低作用与明显的全身血管阻力下降及心输出量和心率增加有关.血浆中去甲肾上腺素水平的变化通常与从静息至站立的体位的改变有关,在滴注本品过程中,该去甲肾上腺素水平的变化显得迟钝,这与交感抑制作用是一致的.24例因癌症,艾滋病,神经病,脊髓损伤和幻觉肢体痛引起的严重顽固性疼痛患者_2,对其他的镇痛方法(包括椎管内注射吗啡或其他麻醉剂)治疗无效.给予0.2～21肛g?h(70kg)的本品后,超过2/3的患者衡量疼痛的指标下降50%或更多,一些患者完全摆脱了吗啡等麻醉剂的应用.6.2Ⅱ期临床试验31例慢性疼痛男性患者包括20例晚期癌症和艾滋病患者和11例脊柱损伤,丘脑性疼痛,臂丛撕裂及后遗神经痛患者,用包括鞘内途径的阿片类药物治疗疼痛都得不到足够的缓解_4一.鞘内持续滴注本品,速度从0.3ng?kg?h增加至疼痛缓解或出现不能耐受的不良反应,最大剂量为300ng?kg?h～.滴定中每隔4h进行一次疼痛评价.利用疼痛强度的形象化模拟评分法(V ASPI),疼痛缓解的形象化模拟评分法(V ASPR)中国新药杂志2005年第14卷第4期和对患者的满意度询问对药效进行评估.疼痛能得到满意缓解的患者继续接受更长时间的治疗方案. 完成试验的24例患者中有19例的V ASPI值降低的平均值是43%,15例患者可减少同时使用的阿片类药物50%的用量.最常见的不良反应有眼球震颤,精神错乱,找词困难,恶心,眩晕,头痛及步态和平衡障碍等,当给药剂量降低或停药后,不良反应可逆.有9例患者给药长达9个月.1例具有23年慢性顽固性传入神经阻滞疼痛和患肢痛患者,在接受持续恒速滴注2.0ng?kg?h的本品治疗后,痛觉过敏和超敏完全消失,疼痛得到完全缓解Z22一. 在对急性术后患者进行镇痛作用和安全性研究中,进行选择性手术的30例患者中有腹式子宫切除术者6例,根治性前列腺切除术者7例和髋部完全替换术者l7例引.于术前开始至术后的48～72h分别向12例和6例患者持续鞘内滴注本品0.7和7tLg?h一,其余的给予安慰剂.可用于药效评价的26例患者的结果表明,本品组的吗啡等效消耗量与安慰剂组比较在24～48h内有显着性差异;术后8h 内本品组的V ASPI值与安慰剂组比有明显降低.6 例高剂量组中的4例出现眩晕,视力模糊,眼球震颤和镇静作用等不良反应,停药后消失.考虑到不良反应,0,7tLg?h的剂量可能比7tLg?h更为理想.6.3Ⅲ期临床试验在开放性试验中,9例只有很短期望寿命的癌症晚期患者使用1～100ng?kgI.h的本品后疼痛得到缓解,6.5周内无耐受性形成_l一.260例恶性神经性疼痛患者的安慰剂对照试验中,102例患者的中期结果分析显示:与安慰剂比较,使用本品后疼痛分数下降40%_l.240例患有阿片类耐受的非恶性(主要源自神经性)疼痛患者中有162例接受本品治疗,78例给予安慰剂.与安慰剂比较本品产生显着的镇痛作用;对鞘内注射吗啡无效的顽固性疼痛患者,对本品也有显着的镇痛反应.根据患者主观评估,本品组和安慰剂组分别有43%和18%的患者得到中等至完全的缓解¨J.治疗1l1例(24～85岁)癌症或艾滋病患者的顽固性疼痛的Ⅲ期临床试验采用双盲和安慰剂对照法,于1996年3月12日一1998年7月11日在美国,澳洲和荷兰的32个研究中心进行|8?.受试者要求V ASPI值≥50,按2:1的比例随机分成2组,分别接受本品和安慰剂的治疗.所有患者的给药时间限制在2周内.首先是鞘内滴定本品或安慰剂共5～6d,有应答者接着给药5d维持治疗;无应答者则一493—ChineseJournalofNewDrugs2005,V ol14No.4交换到对应的治疗组.两组患者均以阿片类药物作基础治疗,本品和安慰剂组的口服吗啡等效消耗剂量分别为300和600mg?d,其中有36例使用鞘内吗啡给药.试验的可评价病例为108例.在招募的初期阶段给药剂量为5ng?kg?h,以后改为0.4/,g-h～.每12h增加至最大耐受剂量.根据前48例招募者的安全性评价结果将以后60例的受试者的起始剂量改为0.1Fg?h或更低,每隔24h增加至产生镇痛作用或至最大剂量2.4Fg?h～.给药前本品和安慰剂组的V ASPI平均值分别是73.6和77.9.给药后分别改善53.1%和18.1%(P<0.001),疼痛得到中等至完全缓解的分别占52.9% 和17.5%(P<0.001).有5例患者在给予本品后疼痛得到完全缓解.分别有50%和17.5%(P=0.001)的患者对本品和安慰剂治疗有反应.试验证明本品和安慰剂组疗效有显着性差异.不良反应多与剂量相关,包括晕眩,眼震颤,精神错乱,异常步态,嗜睡,发热,体位性低血压,尿潴留或尿失禁,恶心和呕吐等,这些不良反应易识别且为可逆,发生率随初始剂量和滴定频率的降低而下降.按FDA的要求重新设计的Ⅲ期治疗严重慢性疼痛的临床试验于2002年8月一2003年6月进行,采用随机双盲,安慰剂对照的方法.220例18岁以上用其他方法得不到缓解的严重慢性疼痛患者接受试验.本品在3周内剂量缓慢增加,试验证明了本品在更低剂量下和在更缓慢的滴注速度条件下的安全性和有效性.在此次的临床试验中,几乎没有严重的不良反应发生,其发生率与安慰剂组相当~24--26].另外,据报道在1999年12月完成的2个临床试验中,超过700例顽固性疼痛患者接受鞘内给药, 包括对吗啡治疗抵抗或对其不良反应不能耐受的患者,证明该药安全且有很好的长达3年的耐受性_1J. 在120个临床中心的9个临床对照试验中有超过1000例慢性疼痛患者接受过本品鞘内给药.也证实了本品鞘内途径在急慢性疼痛中的镇痛作用_6. 7适应证及给药途径适应证是严重慢性疼痛.该药将首先定位于3种人群,包括患有慢性疼痛的癌症和艾滋病患者,以及神经性疼痛患者_2;特别适用于对传统治疗包括鸦片类药物治疗无效的顽固性疼痛和对传统治疗药物不能耐受的疼痛.本品的临床推荐给药途径是鞘内注射或称椎管内注射,包括硬膜下和蛛网膜下隙注射,即通过埋植.--——494.--——中国新药杂志2005年第14卷第4期在皮下的泵直接将药物泵人脊髓周围的脑脊液中. 该给药途径可降低给药剂量,减少全身给药的不良反应,且选择性地将药物转运至中枢神经系统作用部位….8药物不良反应在临床试验中发现鞘内注射本品产生的不良反应主要有_1'2l_:①心血管反应:心率缓慢和直立性低血压,呈剂量依赖性.②中枢神经系统反应:眩晕,眼球震颤,共济失调,兴奋,幻觉,镇静和昏迷等均有报道.其中一些不良反应在停药后或减少剂量后需几天至几个星期才可清除.③胃肠道反应如恶心,呕吐和腹泻等.④其他如皮疹,结膜充血,鼻充血,肝功能异常和尿潴留等.当受试者降低滴注速率或者停止滴注,这些不良反应会减弱或消失.主要不良反应发生于剂量0.2～5.3Fg?h一[.9结语1998年,本品的原研发公司美国Neurex公司以7.41亿美元的证券交易价值将其转让给Elan公司_2.2001年7月9日,本品被欧盟药品评审委员会(EMEA)批准为孤儿药(源自EMEA网站). 2003年5月,Elan公司向EMEA提交上市申请. 2004年1月7日,Elan公司获得本品的Ⅲ期临床评价的主要试验终点(primaryendpoint).2004年6月28日Elan公司向FDA提交有关新药上市许可申请(NDA)的修正案2,从2004年第一季度开始,在新药上市许可的审评期间,本品(商品名Prialt)以TreatmentIND(TreatmentInvestigationalNewDrugs,研究中的新药用于治疗)的形式已在美国一些选定的疼痛中心在一定限度上使用,用于缓解用其他方法治疗无效的疼痛_2.Ziconotide作为一种非吗啡类镇痛药,将给慢性疼痛这一有意义且尚空白的治疗领域带来福音.[作者简介]陈时宏(1971一),女,主管药师,主要从事神经系统药物研究.联系电话:(0592)5977888[参考文献]～一瑚㈣¨卜一¨一一一__c一l_～一_堇_三_詈_毫州～～一～一一一～一一_蓍r=¨～～～一～一～～一～一ChineseJournalofNewDrugs2005,V ol14No.4[4]BowersoxSS,TichN,MayoM,eta1.SNX一111:antinociceptive agentN—typevoltage—sensitivecalciumchannelblocker[J一.[17]DrugsFutu,1998,23(2):152160.[5]NadasdiL,Y amashiroD,ChungD,etalStructureactivityanal—ysisofaConuspeptideblockerofN—-typeneuronalcalciumchan—-nels[J]Biochemistry,1995,34(25):80768081[6][7[8][9][1O][12][13][14][15[16]WermelingD,DrassM,EllisD,etaPharmacokineticsand pharmacodynamicsofintrathecalziconotideinchronicpainpa—tients[J].JClinPharmacol,2003,43(6):624—636 BowersoxSS,LutherRPharmacotherapeuticpotentialofomega—conotoxinMVⅡA(SNX111),anN—typeneuronalcalcium channelblockerfoundinthevenomofConusmagus[J].),_con,1998,36(11):16511658StaatsPS,Y earwoodT,CharapataSG,etalIntrathecalzi—conotideinthetreatmentofrefractorypaininpatientswithcan—cerorAIDS:arandomizedcontrolledtrial[J].JAMA,2004,291 (1):63—70.BowersoxSS.SinghT,NadasdiL,eta1.Cardi0vasculareffectsof omegaconopeptidesinconsciousrats:mechanismsofaction[JjJ CardiuvascPharmacol,1992,20(5):756—764FoxJA.IrreversibleandreversibleblockadeofIMR32calcium channelcurrentsbysyntheticMVⅡAandiodinatedMVⅡC omega-conopeptides[J].PJlugersAre'h,1995,429(6):873875.MalmbergAB,Y akshTLV oltage—sensitivecalciumchannelsin spinalnociceptiveprocessing:blockadeofN—andPtypechannels inhibitsformalin—inducednociception[J].JNeurusci,1994,14 (8):4882—4890MalmbergAB.Y akshTL.Effectofcontinuousintrathecalinfu—sionofomegaconopeptides,N—typecalcium—channelblockers,on behaviorandantinociceptionintheformalinandhotplatetestsinrats[J].Pain,1995,60(1):83—90BowersoxSS,GadboisT,SinghT,eta1.SelectiveNtypeneuronalvoltage—sensiti~ecalciumchannelblocker,SNX111,pro—ducesspinalantinociceptioninratmodelsofacute,persistentand neuropathlcpain[J].JPharmacolExpTher,1996,279(3): 1243—1249.WangYX,GaoD,PettusM,etalInteractionsofintrathecally administeredziconotide.aselecti,feblockerofneuronalN—type voltage—sensitivecalciumchannels,withmorphineonnociception inrats[J].Pain,2000,84(23):271281.WhiteDM.CousinsMJEffectofsubcutaneousadministrationof calciumchannelblockersonnervei~ury—inducedhyperalgesia [J].BrainRes,1998,8O1(1—2):50—58.XlaoWH,BennettGJ.Syntheticomega—conopeptidesappliedto thesiteofnerveinjurysuppressneuropathicpainsinrats[J]J——-+——*+++-+-++++++一+[18][19][2O][21][22][23][24][25][26][27]中国新药杂志2005年第14卷第4期PharmacolExpTher,1995,274(2):666—672.WangYX,PettusM,GaoD,eta1.Effectsofintrathecaladmin—istrationofziconotide,aselectiveneuronalN—typecalciumchan—nelblocker,onmechanicalallodyniaandheathyperalgesiainaratmodelofpostoperativepain[J].Pain,2000,84(2—3):151158.BowersoxS,MandemaJ,Tarczy—HornochK,eta1.Pharmacoki—neticsofSNX111,aselectiveN—typecalciumchannelblocker, inratsandcynomolgusmonkeys[J].Drug[VletabDispos,1997,25(3):379383McGulreD,BowersoxS,FellmannJD,eta1.Sympatholysisafter neuron—specific,N—type,voltage—sensitivecalciumchannelblock—ade:firstdemonstrationofN—channelfunctioninhum?。

丹参总酚酸联合anti-PD-L1调控髓源性巨噬细胞浸润抑制乳腺癌发生发展

网络出版时间：２０２３－０９－２７１７：０９：２７　网络出版地址：ｈｔｔｐｓ：／／ｌｉｎｋ．ｃｎｋｉ．ｎｅｔ／ｕｒｌｉｄ／３４．１０８６．Ｒ．２０２３０９２６．１４３４．０３０丹参总酚酸联合ａｎｔｉＰＤＬ１调控髓源性巨噬细胞浸润抑制乳腺癌发生发展宋梦瑶１，钱　程１，陆　茵１，２（１．南京中医药大学药学院，江苏省中药药效与安全性评价重点实验室，２．江苏省中医药防治肿瘤协同创新中心，江苏南京　２１００２３）收稿日期：２０２３－０４－１１，修回日期：２０２３－０７－２２基金项目：国家自然科学基金资助项目（Ｎｏ８１９７３７３４）；江苏省研究生创新项目（ＮｏＫＹＣＸ２１１７９４）作者简介：宋梦瑶（１９９８－），女，硕士生，研究方向：中药抗肿瘤药理，Ｅｍａｉｌ：２０２００６６２＠ｎｊｕｃｍ．ｅｄｕ．ｃｎ；陆　茵（１９６３－），女，博士，教授，博士生导师，研究方向：活血化瘀中药对肿瘤发生发展的影响，通信作者，Ｅｍａｉｌ：ｌｕｙｉｎｇｒｅｅｎ＠ｎｊｕｃｍ．ｅｄｕ．ｃｎｄｏｉ：１０．１２３６０／ＣＰＢ２０２３０２００４文献标志码：Ａ文章编号：１００１－１９７８（２０２３）１０－１８８４－０７中国图书分类号：Ｒ３３２；Ｒ２８２７１；Ｒ３２９２４；Ｒ３９２１２；Ｒ７３７９摘要：目的　探究丹参总酚酸（ｔｏｔａｌｓａｌｖｉａｎｏｌｉｃａｃｉｄ，ＴＳＡ）联合ａｎｔｉＰＤＬ１通过调控髓源性巨噬细胞瘤内浸润抑制乳腺癌的发生发展。

方法　构建Ｅ０７７１乳腺癌皮下肿瘤模型。

２５只小鼠随机分为空白组、模型组、ＴＳＡ组（ＴＳＡ１０ｇ·ｋｇ－１），ａｎｔｉＰＤＬ１组（ａｎｔｉＰＤＬ１１０ｍｇ·ｋｇ－１），ＴＳＡ联合ａｎｔｉＰＤＬ１组（ＴＳＡ１０ｇ·ｋｇ－１＋ａｎｔｉＰＤＬ１１０ｍｇ·ｋｇ－１）。

ＴＳＡ组每日灌胃给药，ａｎｔｉＰＤＬ１每３天１次腹腔注射，连续给药１４ｄ。

半乳糖化阿昔洛韦三节前药纳米给药系统的研究

Conclution: The results of the study indicated that the preparation of acyclovir for the
triparted pre-drug and then prepared for nanoparticle whose surface coupled with glycosyl as a drug delivery systems(TDDS),Which could enhance the uptake of drugs into liver,was an effective strategy and a new approach for the treatment of hepatitisB.
Spectrophotometer,FTIR,TEM,LPA(laser particle analyzer).The cytotoxicity of GANP on
i
广东药学院硕士研究生学位论文
human liver cancer cells HepG-2 compared with ANP and ACV solution group was studied using MTT methord,The liver target of GANP and ANP were investigated after intraperitoneal injection in mice,Compared with ACV solution injection.
方法：采用正交设计对筛选出的活化条件进行优化，以 SACV 活化率为指标，
考察并优化活化时间、活化温度、 NHS 与 SACV 及 DCC 与 SACV 摩尔比等反应条件；以 BSA-ACV 偶联率为评价指标，以 BSA 溶液浓度、pH、反应时间、摩尔比为考察因素，筛选最佳反应条件。采用 HPLC 法测定 BSA-ACV 在不同 pH 值的缓冲溶液中的稳定性，根据缓冲溶液中游离的阿昔洛韦浓度间接计算阿昔洛韦三节前药的浓度，进行拟合计算，数学拟合求算降解动力学方程。以去溶剂法制备 BSA-ACV 纳米粒，对 BSA-ACV 溶液浓度、溶液 pH、乙醇用量及固化剂用量等制备条件进行了优化以得到最佳的形态、粒径，确定制备 BSA-ACV 纳米粒的最佳工艺条件，纳米粒表面偶联半乳糖苷，并用傅立叶红外光谱分析仪、紫外-可见分光光度计、差热分析、透射电子显微镜、激光粒度分布仪对产物予以分析表征。以阿昔洛韦三节前药纳米粒（ANP）及阿昔洛韦溶液为对照组，采用 MTT 法测定半乳糖化阿昔洛韦三节前药纳米粒（GANP）对人肝癌细胞 HepG-2 的毒性作用；以小鼠腹腔注射方式给药，以阿昔洛韦溶液制剂为对照，研究了半乳糖化阿昔洛韦三节前药纳米粒及阿昔洛韦三节前药纳米粒在小鼠体内肝靶向性。