外源Smad7基因对原代肝星状细胞活性和基因表达调控的影响

目的研究外源Smad7基因对原代肝星状细胞（HSC）活性和基因表达调控的影响。方法采用胶原酶原位灌注、密度梯度离心法分离SD大鼠原代HSC，并予转化生长因子口。（TGFβ1）刺激，同时分别以重组腺病毒AdSmad7和对照病毒AdGFP感染原代HSC，RT—PCR法检测TGFβ1、Smad3 mRNA、Smad7 mRNA在HSC中的表达，免疫细胞化学法检测HSC中a一平滑肌肌动蛋白（a—SMA）和Smad7表达。结果RT—PCR显示，与TGFβ1对照组比较，AdSmad7组Smad7 mRNA表达显著上调（P〈0．05），但TGFβ1、和Smad3 mRNA表达差异无统计学意义。免疫细胞化学染色显示，与其他各组比较，AdSmad7组HSC胞质中Smad7表达最高，α—SMA表达则明显降低（P〈0．01）。结论重组复制缺陷型腺病毒AdSmad7可在HSC中高效表达；外源Smad7基因可阻断TGFβ1，对HSC的活化作用，但对Smad3和TGFβ1，mRNA表达无明显影响。     

虫草菌丝和丹参对肝星状细胞活化、Ⅰ型胶原及转化生长因子β1mRNA与蛋白表达的影响

目的探讨虫草菌丝、丹参抗肝纤维化的作用机理。方法虫草菌丝与丹参流浸膏分别给大鼠经口灌胃给药后分离药物血清,观察药物血清对体外传代活化的大鼠肝星状细胞α－平滑肌肌动蛋白（SMactin,SMA）表达、[3H]TdR及[3H]脯氨酸掺入,TGFβ1、I型前胶原mRNA表达及其蛋白生成量的影响。结果丹参抑制HSC的SMA表达、I型胶原生成量及细胞内[3H]脯氨酸掺入的作用显著,分别为对照组的39.8％,42．7％与38．9％（P＜0．01;前2项显著低于虫草菌丝组,P＜0．05）,尚可抑制细胞1型前胶原＜mRNA,TGFβ1＜mRNA及其蛋白的表达（P＜0．01）;虫草菌丝对上述指标也均有抑制作用,以抑制TGFβ1＜mRNA及其蛋白表达的作用尤著,分别为对照组的47．7％和21．1%(P＜0．01）;显著低于丹参组,P＜0．05。结论丹参具有较强的抑制HSC活化与胶原合成的作用,抑制胶原合成主要作用于前胶原转录后的水平;虫草菌丝的主要作用点抑制HSC的TGFβ1mRNA表达与自分泌。     

重组转化生长因子β3基因对大鼠肝星状细胞Ⅰ型胶原表达的影响

目的观察转化生长因子D3基因（TGFβ3）对大鼠肝星状细胞株（HSC—T6）Ⅰ型胶原合成的影响。方法TGFβ3表达质粒[pcDNA3．1（＋）-TGFβ31和TGFβ1表达质粒[pcDNA3．1（＋）-TGFD11的构建。通过脂质体介导方法，将pcDNA3．1（＋）-TGFβ1、pcDNA3．1（＋）-TGFβ3分别及共同转染体外培养的HSC—T6细胞，荧光定量PCR法及Westernblot法分别检测转染后TGFβ1、TGFD3、Ⅰ型胶原mRNA及蛋白质的表达。将pcDNA3．1（＋）-TGFD1转染HSC—T6细胞，经G418筛选建立高表达TGFD1的HSC～T6细胞克隆，pcDNA3．1（＋）-TGFD3转染克隆细胞，荧光定量PCR法检测转染后TGFβ3、TGFβ1及Ⅰ型胶原mRNA的表达，Westernblot法检测TGFβ1、Ⅰ型胶原蛋白的表达情况。结果构建的pcDNA3．1（＋）TGFD3、pcDNA3．1（＋）-TGFD1质粒可转染HSC—T6细胞，转染率28．2％。pcDNA3．1（＋）TGF侈3转染细胞后，Ⅰ型胶原mRNA及蛋白的表达较空白组及对照组增加，以72h增高最为明显（P〈0．05）；共转染组Ⅰ型胶原mRNA及蛋白质的表达较pcDNA3．1（＋）-TGFβ1转染组明显降低（P〈0．05）。TGF侈3转染克隆细胞后，TGFD1mRNA表达较克隆组无明显改变（P〉0．05），而蛋白质表达明显下降（P〈0．05），Ⅰ型胶原mRNA及蛋白质表达均较克隆组明显降低（P〈0．05）。结论TGFD3基因转染正常培养的HSC—T6细胞，增加Ⅰ型胶原的表达；转染高表达TGFβ1的克隆组HSC—T6细胞，Ⅰ型胶原表达明显降低，提示TGFβ3对肝纤维化的发生有抑制作用。     

抗纤二号药物血清对活化型肝星状细胞的影响

为了解抗纤二号的抗肝纤维化作用机制，采用动物体内给药，分离药物血清，作用于体外培养细胞的血清药理学实验方法，探讨该方对肝星状细胞（HSC）激活的标志平滑肌肌动蛋白（smooth muscle actin，SMA）表达的影响及Ⅰ、Ⅲ型胶原和转化生长因子β1（TGFβ1）及其下游信号Smad 3表达的影响，从细胞分子学水平探讨该方抗肝纤维化的作用机制。     

甘草酸对转化生长因子β1刺激肝星状细胞信号传导的作用

目的探讨甘草酸对转化生长因子β1(TGF β1)刺激大鼠肝星状细胞(HSC)胞内信号传导通路的作用。方法体外分离、培养大鼠肝HSC,甘草酸与TGFβ1刺激的HSC共同孵育。逆转录聚合酶链反应法检测1-1000 μmol/L甘草酸对TGF β1刺激的HSC中Smad2、3、7 mRNA的表达;Western印迹法检测1-1000μmol/L甘草酸对TGF β1刺激的HSC中Smad2、3、7蛋白及Ⅰ、Ⅲ型胶原蛋白的表达水平。结果TGFβ1促进HSC中Smad2、3、7 mRNA及蛋白的表达,同时促进Ⅰ、Ⅲ型胶原蛋白的表达。1-1000μmol/L甘草酸抑制Smad2、3、7 mRNA及蛋白的表达,并且抑制Ⅰ、Ⅲ型胶原蛋白的表达,均呈剂量依赖性。结论甘草酸可能通过干预大鼠HSC中TGFβ信号通路而减少胶原合成和发挥抗纤维化作用。     

Smad3、Smad7基因表达与肝纤维化发病关系研究

目的：探讨Smad3和Smad7编码基因在肝纤维化大鼠肝星状细胞（HSC）中的表达及意义。方法：注射四氯化碳（CCl4)制备大鼠肝纤维模型，按诱导时间将动物随机分为1、4、8周及正常对照组4例，行肝组织胶原VG染色，转化生产因子（TGF）β1免疫组化检测，同时分别以RT－PCR、Western blot检测原工分离HSC的Smad3、Smad7mRNA和蛋白表达。结果：肝纤维化形成过程中肝组织胶原生成逐渐增多，TGFβ表达增强。与正常对照相比，各期肝纤维化大HSC中Smad3mRNA表达增加（P＜0．05），注射CCl4后1周，Smad7mRNA较正常一过性升高（P＜0．05）。第4周和第8周表达水平则持续下降（P＜0．01），Smad3和Smad7的蛋白表达与其mRNA基本一致。结论：Smad3和Smad7在肝纤维化发生，发展中起不同的介导作用，提示通过靶向性阻断HSC Smad3信号和（或）加强Smad信号有望成为治疗肝纤维化的新手段。     

Ciglitazone抑制人肝星状细胞外基质蛋白表达的分子机制

目的探讨过氧化物酶体增殖物激活受体（PPARγ）配体是否通过阻断肝星状细胞的TGF β1型受体（TGF βR1）信号途径，阻止Smad3磷酸化而抑制纤溶酶原激活物抑制剂1（PAI-1）、胶原蛋白1αI的表达，发挥其抗纤维化的作用。方法用促进脂肪细胞分化的培养液预处理人肝星状细胞LX-2，使其呈现脂肪细胞的某些表型，从活化状态转为静止型，分别加用TGF β1或TGF β1加TGF βR1激酶抑制剂SB431542或TGF β1加PPAR γ配体Ciglitazone处理LX-2细胞。分别用荧光实时定量PCR、Western blot、荧光素酶分析的方法检测PPAR γ配体对LX-2细胞用TGF β1诱导前后Smad3磷酸化水平、PAI-1的mRNA与蛋白表达水平和PAI-1启动子活性、胶原蛋白1αI mRNA表达水平的影响。结果诱导脂肪细胞分化的培养液可使LX-2细胞出现脂质沉积与静止型肝星状细胞的标志物之一PPAR γ表达的增加。TGFD1以剂量和时间依赖的方式增加细胞外基质蛋白的表达，胶原蛋白1aI和PAI-1 mRNA的表达在3h内增加3倍，PAI-1蛋白的表达在6h内增加8倍。TGF β1诱导的Smad3磷酸化导致胶原蛋白1aI和PAI-1表达增加。PPARγ配体Ciglitazone与TGF βR1激酶抑制剂SB431542，均以剂量依赖的方式阻断TGF β1的上述作用，10μmol／L SB431542、10μmol／L的Ciglitazone可以阻断TGF β1诱导的Smad3磷酸化、胶原蛋白1αI mRNA、PAI-1 mRNA和蛋白的表达。结论PPAR γ配体Ciglitazone抗纤维化作用可能与其对TGF β1-TGF βR1信号传导、Smad3磷酸化的阻断作用有关，进而抑制胶原蛋白1αI和PAI-1的表达。     

特异性小干扰RNA对晚期糖基化终产物受体介导肝星状细胞激活和胶原生成的抑制作用

目的 探讨特异性小分子干扰RNA对晚期糖基化终产物受体（RAGE）介导肝星状细胞（HSC）激活和胶原生成的影响。方法 构建RAGE特异性siRNA表达载体，经脂质体转染入HSC—T6细胞，以空白和转染非特异性siRNA表达载体pGCsi-C为对照，分别用实时荧光定量PCR和Western印迹法检测各组HSC—T6细胞RAGE、α平滑肌肌动蛋白（α—SMA）和Ⅰ型胶原基因及蛋白的表达。结果 与空白对照组和pGCsi—C组相比，转染特异性siRNA表达载体pGCsi—R1的HSC—T6细胞RAGE mRNA和相对分子质量50×10^3、46×10^3的RAGE蛋白表达分别下调79．65％、78．04％（F=26．005，P〈0．01），56．09％、54．93％（F=365．185，P〈0．01）和63．67％、59．67％（F=386．07，P〈0．01），同时α—SMAmRNA和蛋白、Ⅰ型胶原mRNA和蛋白的表达也受到明显抑制，分别为空白对照组和pGCsi—C组的57．53％、65．50％（F=20．913，P〈0．05）、58．48％、62．80％（F=56．592，P〈0．05）、71．16％、74．23％（F=18．091，P〈0．05）和71．91％、76．09％（F=17．299，P〈0．05）。结论 RAGE特异性siRNA可在细胞内稳定表达，并能有效抑制HSC的激活和Ⅰ型胶原生成。     

丹参酚酸B盐对转化生长因子β1活化的大鼠肝星状细胞内p38丝裂原活化蛋白激酶通路及其转录因子的影响

目的 探讨丹参酚酸B盐(SA-B)对转化生长因子β1(TGFβ1)活化的大鼠肝星状细胞(HSC)内p38丝裂原活化蛋白激酶(MAPK)信号传导通路的影响. 方法 分离并培养正常大鼠HSC,将TGFβ1和SA-B直接添加于原代HSC的无血清培养液中,用p38信号通路特异性阻断剂SB203580和细胞外信号调节激酶(ERK)信号通路特异性阻断剂PD98059分别阻断HSC内p38MAPK和ERK信号通路.细胞内总的P38蛋白、MKK3/6蛋白及MEF2A、MEF2C的测定分为空白对照组、SA-B组、SA-B+ TGF β1组和TGFβ1组;磷酸化P38蛋白、MKK3/6蛋白和α-SMA蛋白的测定分为空白对照组、SA-B组、SA-B+ TGF β1组、TGFβ1组、PD98059、PD98059+ SA-B组、PD98059+ TGFβ1组和SA-B+ PD98059+ TGFβ1组;SA-B对TGFβ1刺激的HSC内MEF2和Ⅰ型胶原报道基因的影响分为突变型(mt)对照组、野生型(wt)对照组、TGFβ1组、SA-B+ TGF β1组、SA-B组、SB203580+ TGF β1组、SB203580组.Western blot法检测HSC内磷酸化和总P38蛋白、MAPK激酶3/6 (MKK3/6)蛋白、肌细胞增强因子2(MEF2)A、MEF2C、α-平滑肌肌动蛋白(α-SMA)的表达;荧光素酶报道基因测定法检测MEF2报道基因和Ⅰ型胶原启动子的活性.多组间数据的多重比较用q检验.结果 SA-B组磷酸化P38蛋白相对表达量为0.33±0.05,明显低于空白对照组(q=7.08,P＜0.01); SA-B +TGF β1组的磷酸化P38蛋白相对表达量为0.46±0.04,明显低于TGFβ1组(q=10.45,P＜0.01);SA-B组磷酸化MKK3/6蛋白相对表达量为0.11±0.07,明显低于空白对照组(q=3.944,P＜0.05);SA-B+ TGF β1组磷酸化MKK3/6蛋白相对表达量为0.28±0.07,明显低于TGFβ1组(q=7.91,P＜0.01);SA-B+ TGFβ1组和SB203580+ TGF β1组MEF2报道基因的相对荧光素酶活性分别为2.93±0.09和2.50±0.05,均明显低于TGFβ1组(q值分别为35.35和37.2,P值均＜0.01);SA-B组MEF2C及MEF2A的相对表达量分别为15.82±0.97和13.00±0.40,均明显低于空白对照组(q值分别为5.18和13.32,P值均＜0.01);SA-B+ TGF β1组MEF2C及MEF2A的相对表达量分别为13.40±0.72和20.47±0.83,均明显低于TGF β1组(q值分别为43.93和12.52,P值均＜0.01); SA-B+ TGFβ1组α-SMA相对表达量为8.76±0.44,明显低于TGFβ1组(q=20.35,P＜0.01); SA-B+ SB203580+TGF β1组α-SMA相对表达量仅为3.57±0.49,明显低于TGFβ1组(q=39.78,P＜0.01);SA-B+ TGF β1组和SB203580+ TGF β1组Ⅰ型胶原报道基因的相对荧光素酶活性分别为1.61±0.05和1.42±0.07,较TGFβ1组明显降低(q值分别为26.4和27.62,P值均＜0.01).结论 SA-B可能通过抑制原代HSC内TGFβ 1的p38MAPK信号传导通路,抑制HSC的活化.     

反义转化生长因子βⅠ型受体表达质粒对大鼠肝星状细胞功能的影响

目的　观察反义转化生长因子 (TGF) βⅠ型受体 (TβRⅠ )表达质粒对大鼠肝星状细胞 (HSC)增殖及细胞外基质分泌的影响。方法　双酶灌注和梯度离心法分离大鼠HSC ,将构建的反义TβRⅠ真核细胞表达质粒与 pcDNA3空质粒经脂质体转染培养活化的HSC ,通过RT PCR、Western印迹检测外源导入质粒在HSC中的表达 ,采用MTT法、3 H TdR掺入法检测细胞增殖情况 ,ELISA法检测TGF β1含量变化 ,并应用Western印迹检测HSC中Ⅰ、Ⅲ型胶原表达。结果　反义TβRⅠ真核细胞表达质粒可抑制活化HSC中TβRⅠmRNA及蛋白表达。与 pcDNA3转染组相比 ,反义TβRⅠ质粒表达可抑制HSC增殖 (P <0 .0 1) ,降低TGF β1含量 (P <0 .0 5 ) ,抑制Ⅰ、Ⅲ型胶原分泌 (P <0 .0 5 )。结论　重组反义TβRⅠ表达质粒可在HSC中获得较好表达 ,并可显著抑制HSC增殖及细胞外基质分泌。     

Inhibitory effect of human interferon-beta-1a on activated rat and human hepatic stellate cells

Background and Aims: Hepatic stellate cells (HSC) are the primary cell type mediating hepatic fibrosis. Although known for its antiviral effects, the inhibitory effects of interferon‐beta (IFN‐β) on HSC treatment have not yet been established. Methods: Both human and rat activated HSC cell lines were incubated with increasing concentrations of recombinant human IFN‐β1a (rhIFN‐β1a) for 24, 48 or 72 h. The effects of rhIFN‐β1a on α‐smooth muscle actin (α‐SMA), collagen types I and III, transforming growth factor‐β1 (TGF‐β1), platelet‐derived growth factor‐BB (PDGF‐BB), and mothers against decapentaplegic homolog (Smad4, Smad7) expression in HSC were examined using Western blotting and immunocytochemistry. Proliferation of HSC was evaluated via bromodeoxyuridine assay. Results: rhIFN‐β1a treatment had a dose‐dependent, inhibitory effect on α‐SMA and collagen type I protein expression. In addition, rhIFN‐β1a decreased the expression of collagen type III, TGF‐β1, PDGF‐BB and Smad4 protein expression in HSC compared with untreated cells. We also observed increased Smad7 protein expression and decreased proliferation in rhIFN‐β1a‐treated HSC. Conclusions: Our data suggest that rhIFN‐β1a treatment decreased α‐SMA and collagen expression and inhibited the activation of HSC through the inhibition of the TGF‐β and PDGF pathways.     

Down-regulation of transforming growth factor β1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells

AIM： To investigate the effect of herbal compound 861 （Cpd861） on the transforming growth factor-β1 （TGFβ1）/ activin receptor-like kinase 1 （ALK1, type Ⅰ receptor） signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells.
METHODS： LX-2 cells were treated with TGFβ1 （5 ng/mL） Cpd861 （0.1 mg/mL）, TGFβ1 （5 ng/mL） plus Cpd861 （5 ng/mL） for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA （α-smooth muscle actin）, ALK1, Id1 （inhibitor of differentiation 1）. Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of α-SMA.
RESULTS： In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathway- related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h.
CONCLUSION： Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.     

转化生长因子β1对不同活化状态肝星状细胞增殖与Ⅰ型胶原表达的影响

目的 观察不同活化状态肝星状细胞(HSC)对外源性转化生长因子-β_1(TGF-β_1)旁分泌刺激的生物学效应作用。方法 原代分离培养大鼠HSC,无包被塑料培养皿上分别培养1、4、7d,细胞处于静止、中间活化与完全活化状态,继以10～500 pmol/L TGF-β_1温育细胞24h,~3H—TdR掺入法测定细胞增殖,western blot法检测细胞α-平滑肌肌动蛋白(α-SMA)与Ⅰ型胶原蛋白表达沉积,~3H-脯氨酸掺入与胶原酶消化法测定细胞总胶原的分泌量。100pmol/L TGF-β_1温育细胞15～90min,northern blot法检测细胞Ⅰ型前胶原mRNA的表达水平。结果 TGF-β_1浓度依赖性抑制培养1d HSC的细胞增殖,10～500 pmol/L TGF-β_1浓度组细胞内~3H—TdR掺入率分别为对照组的52.8％～16.8％,与对照组比较,q值为5.44～10.37,P<0.01。但TGF-β_1对培养4d与7d的细胞增殖无影响。随细胞活化,HSC基础性α-SMA、Ⅰ型胶原蛋白与mRNA水平明显增加,而TGF-β_1刺激各培养时间HSC以上蛋白与基因的表达。培养1、4、7d HSC基础水平与TGF-β_1刺激的总胶原分泌量分别为(804±274)dpm/孔与(1 200±708)dpm/孔;(2 966±1 701)dpm/孔与(6 160±1 123)dpm/孔;(2 580±767)dpm/孔与(4 583±1 467)dpm/孔,后2组组内比较,t值分别为3.84与2.96,P<0.01或P<0.05。以培养4d HSC     

Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats

BACKGROUND & AIMS: Numerous studies implicate transforming growth factor (TGF)-beta signaling in liver fibrogenesis. To perturb the TGF-beta pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF-beta signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs). METHODS: Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary-cultured cells. RESULTS: Smad7-overexpressing BDL rats displayed reduced collagen and alpha-SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)(9)-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF-beta-dependent proliferation inhibition of HSC. Smad7 did not decrease expression of alpha-SMA, but immunofluorescent staining with anti alpha-SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton. CONCLUSIONS: In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation.     

The role of Smad3 in mediating mouse hepatic stellate cell activation.

Transforming growth factor beta (TGF-beta) is the most potent profibrogenic mediator in liver fibrosis. Although Smad proteins have been identified as intracellular mediators in the TGF-beta signaling pathway, the function of individual Smad proteins remains poorly understood. The aim of this study was to explore the contribution of Smad3 in mediating TGF-beta responses in a model of acute liver injury in vivo and in culture-activated hepatic stellate cells (HSCs). Wild-type, Smad3 heterozygous or Smad3 homozygous knockout mice were treated with a single intragastric administration of CCl(4). After 72 hours, the induction of hepatic collagen alpha1(I) and alpha2(I) messenger RNA (mRNA) levels in Smad3 knockout mice was only 42% and 64%, respectively, of the levels induced in wild-type mice. However, smooth muscle alpha-actin (alpha-SMA) was expressed at a slightly higher level in livers from knockout mice compared with wild-type mice. In culture-activated HSCs from Smad3 knockout mice, collagen alpha1(I) mRNA was 73% of wild-type HSCs, but alpha-SMA expression was the same. HSCs from knockout mice showed a higher proliferation rate than wild-type HSCs. Smad3-deficient HSCs did not form TGF-beta1-induced Smad-containing DNA-binding complexes. In conclusion, (1) maximal expression of collagen type I in activated HSCs requires Smad3 in vivo and in culture; (2) Smad3 is not necessary for HSC activation as assessed by alpha-SMA expression; (3) Smad3 is necessary for inhibition of proliferation of HSCs, which might be TGF-beta-dependent; and (4) Smad3 is required for TGF-beta1-mediated Smad-containing DNA-binding complex formation in cultured HSCs.     

肝苏对人肝细胞和肝星状细胞增殖、氧应激以及细胞外基质表达的影响

背景：有研究显示肝苏对肝细胞具有保护、抗炎、抗氧化和抗肝纤维化的作用,但其作用机制未明。目的：探讨肝苏对人肝细胞和肝星状细胞（HSC）增殖、氧应激以及细胞外基质表达的影响。方法：分别用0．01～1．0mg／ml肝苏培养肝细胞和HSC,以M1丫r法检测肝苏对肝细胞和HSC增殖的影响：用次氮基三乙酸铁（Fe-NTa）和0．05—1．0mg／ml肝苏共同培养肝细胞和HSC,检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量;用1．5mg／ml和2．5mg／ml肝苏培养HSC,以酶联免疫吸附测定（ELISA）检测细胞外基质透明质酸（HA）、层黏连蛋白（LN）、I型胶原、Ⅲ型胶原和细胞因子转化生长因子（TGF）-β1含量。结果：在0．05。1．0mg／ml浓度范围内,肝苏可促进肝细胞增殖,但各浓度肝苏对HSC增殖无明显影响。肝苏可增高肝细胞SOD活性,降低肝细胞和HSCMDA含量,但对HSCSOD活性无明显影响。同时肝苏可抑制HSC细胞外基质HA、LN和细胞因子TGF-β1的表达,而I型胶原、Ⅲ型胶原无明显差异。结论：肝苏可促进肝细胞增殖,保护肝细胞和HSC免受氧应激损伤,抑制HSC分泌HA、LN和TGF-β1,提示其具有肝细胞保护、抗氧化和抗肝纤维化作用。     

Rearrangement of the cytoskeletal network induced by platelet-derived growth factor in rat hepatic stellate cells: role of different intracellular signalling pathways

BACKGROUND/AIMS: Cytoskeletal reorganization plays an important role in the regulation of different cell functions, such as proliferation and migration. Since platelet-derived growth factor (PDGF) stimulates both proliferation and chemotaxis of hepatic stellate cells (HSC), we investigated the effects of this cytokine on cytoskeletal components of cultured rat HSC. METHODS/RESULTS: Exposure of HSC to PDGF induced the formation of stress fibres and of a ruffled configuration of the plasma membrane, evaluated by both fluorescence and electron microscopy. These modifications were also induced by exposure to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) and abolished by pretreatment with the PKC inhibitor calphostin C, with the Rho inhibitor C3 exoenzyme and with the intracellular calcium chelator MAPTAM, but not with the PI-3 kinase inhibitor wortmannin or with the mitogen-activated protein kinase kinase inhibitor PD 98059. PDGF induced a translocation of Rho from the cytosol to the membrane which was inhibited by C3 exoenzyme and by calpostin C, and which was also induced by PMA. Moreover, PDGF induced a rearrangement of vinculin which was prevented by C3 exoenzyme and calphostin C. CONCLUSIONS: PDGF-induced cytoskeletal reorganization in HSC is dependent on PKC and Rho, thus suggesting that these two pathways may play an important role in the response of liver to injury.