应用SYBR荧光实时定量RT-PCR法检测BMSCs诱导大鼠肝星状细胞中DR5mRNA表达

目的应用SYBR荧光实时定量RT-PCR法检测骨髓间充质干细胞（BMSCs）对大鼠肝星状细胞（HSCs）的死亡受体5（DR5）mRNA表达的影响，探讨BMSCs诱导HSCs凋亡及其机制。方法采用贴壁筛选法培养、纯化SD大鼠BMSCs，传至第4代使用；大鼠原代HSCs细胞及肝纤维原细胞系冻融后传代使用。应用6孔塑料培养板，建立上下双层细胞共培养体系，常规培养。实验分为3组：（1）实验组：BMSCs与HSCs共培养；（2）空白对照组：HSCs单独培养；（3）阴性对照组：大鼠肝纤维原细胞与HSCs共培养。以上培养体系动态观察24、48、72h，应用流式细胞仪检测HSCs细胞凋亡率，采用SYBRGreenI荧光实时定量RT-PCR法检测，以β-actin基因作为内参，计算各组DR5mRNA的相对表达量。结果在共培养组中，BMSCs促进了HSCs凋亡，与其他两组比较差异有显著统计学意义（P〈O．01），空白对照组与阴性对照组比较无统计学意义（P〉0．05）。实验组BMSCs能明显上调HSCs中DR5mRNA的表达，与空白对照组和阴性对照组比较差异有显著统计学意义（P〈O．01）；空白对照组与阴性对照组DR5mRNA的表达比较无统计学意义（P〉O．05）。结论利用SYBR荧光实时定量RT-PCR法检测BMSCs诱导大鼠肝星状细胞中DR5mRNA表达，为进一步研究BMSCs通过死亡受体途径调控HSCs凋亡以及为BMSCs用于治疗肝纤维化的机制研究提供了理论基础。

Detection of DR5 mRNA expression induced by BMSCs in HSCs using SYBR real-time fluorescence quantitative RT-PCR method

Objective To detect the effects of bone marrow mesenchymal stem cells on the mRNA expression of DR5 in hepatic stellate cells.Methods BMSCs were isolated from bone marrow in rats and grown,propagated in culture flask in the passages 4.HSCs and fibroblast cells were recoveried and activated morphologically.The co-culture of MSCs with HSCs was performed by using a 6-well Transwell membranes.The HSCs were seeded in the lower chamber with BMSCs and fibroblast cells(2×105cells/ml) were seeded onto the Transwell membranes of the inner chamber.Cultures were maintained in HSCs in medium for 24 h,48 h,72 h.Three groups were divided randomly:(1)BMSCs co-culture group.(2)HSCs control group.(3)Fibroblast control group.The apoptosis rates of HSCs were determined by flow cytometry.The mRNA expression of DR5 was evaluated with RT-qPCR.Results The increased apoptosis rates of HSCs proliferation with MSCs culture were significant higher than those in the other two groups at times 24 h,48 h and 72 h(all P<0.01).The mRNA expression of DR5 in BMSCs co-culture group was higher than the HSCs control group and fibroblast control group(P<0.01).And there was no significant difference between the HSCs control group and the fibroblast control group(P>0.05).Conclusion In vitro,BMSCs promote apoptosis and significantly increase the mRNA expression of DR5 in rats activated HSCs.