IGFBPrP1 siRNA诱导的大鼠肝星状细胞凋亡及其机制

IGFBPrP1 siRNA induced apoptosis in rat hepatic stellate cells and its mechanism

目的研究IGFBPrP1 siRNA对大鼠肝星状细胞凋亡的影响,并探讨其可能的机制。方法体外培养大鼠肝星状细胞株(HSC-T6),分别设立:正常对照组,阴性对照组,IGFBPrP1 siRNA转染组。IGFBPrP1 siRNA转染大鼠肝星状细胞,转染不同时间后,用CCK-8试剂盒检测肝星状细胞增殖的变化,AnnexinV/PI双标法流式细胞术检测肝星状细胞凋亡的变化,免疫细胞化学染色法检测P53及Bcl-2蛋白表达的变化。结果 (1)IGFBPrP1 siRNA转染大鼠肝星状细胞不同时间(24、48、72 h)后,转染组细胞增殖受到抑制且凋亡率明显增高,与正常对照组及阴性对照组相比,差异均有统计学意义(P<0.01);(2)IGFBPrP1 siRNA转染大鼠肝星状细胞48 h后,与正常对照组及阴性对照组相比,转染组P53蛋白的表达量显著增高(P<0.01);Bcl-2蛋白的表达明显降低(P<0.01)。结论 IGFBPrP1 siRNA能显著抑制大鼠肝星状细胞增殖且能促进其凋亡;上调P53的表达,下调Bcl-2的表达可能是IGFBPrP1 siRNA诱导大鼠肝星状细胞凋亡的途径之一;推测抑制IGFBPrP1的表达有可能成为治疗肝纤维化的新靶点。

Objective To investigate the effect of inhibiting insulin-like growth factor binding protein related protein1（IGFBPrP1） by chemically synthesized small interfering RNA（siRNA） on apoptosis of HSC-T6 cells and to explore the possible mechanism.Methods HSC-T6 were incubated in vitro and divided into three groups as following： normal control group,negative control group and IGFBPrP1 siRNA transfection group.The IGFBPrP1 siRNA was transfected into HSC-T6 cells.After transfecting with IGFBPrP1 siRNA at different time,cell proliferation was measured by CCK-8 kit and cell apoptosis was detected by flow cytometry with AnnexinV/PI double staining,both P53 and Bcl-2 protein expressions were detected by immunocytochemistry.Results（1） The inhibition ratio were decreased and the apoptotic rates of IGFBPrP1 siRNA transfection groups were increased compared with the normal control group and the negative control group at 24 h,48 h,72 h after transfection（P0.01）;（2） Immunocytochemistry showed that the expression of P53 protein was significantly increased and the expression of Bcl-2 protein was significantly decreased compared with the normal control group and the negative control group at 48 h after transfection（P0.01）.Conclusion IGFBPrP1 siRNA is able to inhibit the cell proliferation significantly and induce apoptosis of HSC-T6 cells effectively.Apoptosis of HSC-T6 cells induced by IGFBPrP1 siRNA may be associated with up-regulation of P53 expression and down-regulation of Bcl-2 expression.RNAi against IGFBPrP1 may provide a new strategy for the treatment of liver fibrosis.