大鼠骨髓间充质干细胞体外诱导肝星状细胞系凋亡

Apoptosis of hepatic stellate cell line induced by rat bone marrow mesenchymal stem cells in vitro

目的研究大鼠骨髓间充质干细胞(MSCs)体外诱导大鼠肝星状细胞(HSCs)凋亡及其机制。方法分离培养MSCs,HSC-T6系及纤维原细胞系冻融后传代使用。用6孔塑料培养板,建立上下双层细胞共培养体系。分3组:空白对照组、阴性对照组和实验组。以上体系培养观察24、48和72 h,于倒置相差显微镜下动态观察HSCs细胞形态;免疫组化法检测HSCsα-SMA表达;WST-8法检测HSCs增殖抑制率;流式细胞仪Annexin-V-FITC/PI双染法和DNA凝胶电泳(DNA Ladder)检测HSCs细胞凋亡;RT-PCR、Western blot检测HSCscaspase-3、BAX基因mRNA和蛋白表达。结果实验组出现明显的DNA Ladder,24 h后HSCs增殖抑制率、凋亡率和caspase-3、BAXmRNA和蛋白表达呈时间依赖性显著高于对照组(P<0.01)。结论 MSCs可在体外抑制HSCs增殖,可能通过旁分泌途径诱导HSCs凋亡,其凋亡发生是通过上调caspase-3、BAX表达发挥作用,本研究支持MSCs通过抑制HSCs产生而抗肝纤维化的机制。

Objective To investigate the effect of rat bone marrow mesenchymal stem cells（MSCs） on apoptosis of hepatic stellate cells（HSCs）.Methods MSCs were isolated from bone marrow in rats.HSCs and fiberoblast cells were recoveried and activated,α-SMA expression in HSCs was evaluated immuno-histochemically.The co-culture of MSCs with HSCs was performed by using a 6-well transwell membranes（24 mm diameter,0.4 μm pore size）.The HSCs were seeded in the lower chamber with MSCs and fiberoblast cells（2×105cells/mL） were seeded onto the transwell membrane of the inner chamber.Cultures were maintained in HSCs in medium for 24 h,48 h and 72 h.Three groups were HSCs control group,fiberoblast control group and MSCs group.At the different pointed time,the inhibitory rate of HSCs proliferation with MSCs co-culture was tested by the method of WST-8,and the rate of apoptosis of HSCs was detected by Annexin-V-FITC/PI and DNA Ladder,and the HSCs in co-culture system was collected and the mRNA expressions of caspase-3 and BAX in HSCs were detected by RT-PCR.Theprotein expressions of caspase-3 and BAX in HSCs was evaluated by Western blot.Results With the co-culture time lasting,MSCs group showed the strip of DNA Ladder,the inhibitory rate of HSCs,the rate of apoptosis of HSCs and the mRNA expression of caspase-3,BAX and caspase-3,BAX protein in MSCs group significantly increased at 24 h.Conclusion MSCs may induce apoptosis of HSCs in vitro by up-regulating the expression levels of caspase-3 and BAX,and thus play important role in the antifibrosis.