Hedgehog-Gli 1信号通路对肝星状细胞增殖和凋亡的调控作用

目的 研究肝星状细胞(HSC)中hedgehog(Hh)信号通路成员Shh、patched、smoothened(Smo)和Gli-1的表达,及Hh通路抑制剂环耙明对HSC-T6细胞株增殖、凋亡及其激活的影响.方法 体外培养HSC株,提取细胞总RNA,用RT-PCR扩增Shh、patched、Smo、Gli-1基因;用环耙明作用HSC-T6后采用四甲基偶氮唑盐法检测其在体外对HSC-T6的抑制情况,流式细胞仪检测其对HSC-T6细胞周期的影响,碘化丙啶和膜联蛋白-Ⅴ双染荧光标记法及提取细胞DNA检测HSC-T6细胞凋亡,荧光定量聚合酶链反应检测Gli-1,转化生长因子β1、血小板衍生生长因子、Bcl-2 mRNA的表达水平.结果 HSC-T6中表达有Hh通路家族成员.环耙明在50、100、150、200,250 μmol/L作用下,A值分别为1.55±0.07、1.19 ±0.05,0.78±0.06、0.48±0.03、0.38±0.04,正常对照组为1.74±0.03,环耙明组与正常对照组相比,F=636.81,P<0.01,差异有统计学意义.流式细胞术测出干预后环耙明G0/G1期细胞所占比例为65.08%±1.50%,正常对照组为55.41%±2.54%,两组比较,t=-8.05,P<0.01.药物干预后提取DNA及碘化丙啶和膜联蛋白-Ⅴ双染荧光标记均检测出HSC-T6细胞凋亡.荧光定量结果表明,经环耙明干预后,HSC-T6细胞中Gli-1、转化生长因子β1、血小板衍生生长因子、Bcl-2 mRNA的目的基因相对表达量分别0.28±0.05、0.13±0.04、0.07±0.04、0.17±0.02,正常对照组为1,各基因表达量较正常对照组均明显降低,t值分别为23.09、31.34、43.87和59.10,P值均<0.01,差异有统计学意义.结论 HSC-T6中存在Hh信号通路,环耙明能抑制HSC增殖及增殖周期,促进活化的HSC凋亡,其抑制HSC活化可能是通过抑制Hh-Gli-1信号通路的结果.

The effects of Hedgehog-Glil signaling pathway on proliferation and apoptosis of hepatic stellate cells

Objective To investigate the effect of Hedgehog-Glil signaling pathway on proliferation,apoptosis and activation of hepatic stellate cells(HSCs)in vitro.Methods The expression of Shh,Smo,Ptc and Gli-1 in HSC-T6 cells was analyzed by RT-PCR.HSC-T6 cells were incubated with various concentration of cyclopamine(0,50,100,150,200,250 μmol/L)for 24 hours,cell viability was checked by MTT colorimetric assay,cell cycle was analyzed by flow cytometry,apoptosis was assayed by agarose electrophoresis of DNA and PI-Annexin V fluorescent staining,and the mRNA levels of Gli-1,TGF beta 1,PDGF and Bcl-2 were quantified by real-time RT-PCR.Results RT-PCR indicated that the components of the Hedgehog-Gli1 signaling pathway were expressed in HSC-T6 cells.MTT assay indicated that cyclopamine inhibited cell viability in a concentration dependant manner (F=636.81,P<0.01).Flow cytometry indicated that cells were piled up at G0/G1 phase in cycloparnine treated cells(65.08%±1.50%) as compared to control cells(55.41%±2.54%, t=-8.05,P<0.01).Cyclopamine treatment resulted in apoptosis as indicated by DNA fragmentation and PI-Annexin V staining.The mRNA levels of Gli-1,TGF beta 1,PDGF and Bcl-2 in cyclopamine treated cells were significantly lower than that in control cells (P<0.01).Conclusion Cyclopamine may inhibit the Hedgehog-Gill signaling,and hence repress proliferation and promote apoptosis in hepatic stellate cells.