【摘要】 目的探讨靶向瘦素(leptin)基因的小干扰RNA(small interfering RNA,siRNA)对leptin表达,肝星状细胞(hepatic stellate cell,HSC)的增殖、凋亡及细胞内钙离子浓度(intracellular Ca2+concentration,[Ca2+]i)的影响,为肝纤维化基因治疗提供可行的方案。方法 HSC分为4组:正常对照组、空载体组、siRNA对照组、leptin-siRNA组。依分组要求分别转染HSC后,采用MTT法和流式细胞技术检测各组细胞的凋亡和增殖;应用免疫细胞化学SP法、Western blot法检测leptin蛋白的表达;Fura-2/AM负载检测[Ca2+]i变化。结果与HSC的正常对照组、空载体组及siRNA对照组相比,leptin-siRNA组可以有效抑制leptin基因的表达和细胞增殖(P<0.05),且不同时段(24、48、72h)细胞抑制率之间有显著差异(P<0.05)。leptin-siRNA组细胞凋亡比例明显增加(P<0.01);[Ca2+]i显著降低(P<0.05)。结论leptin-siRNA可以通过沉默leptin基因表达而抑制HSC的增殖、促进凋亡和减少[Ca2+]i。leptin在肝纤维化发展中起着重要作用,有望成为肝纤维化一个新的预测和预后的标志。更多还原

【Abstract】 Objective To evaluate the effect of silencing leptin by small interfering RNA(siRNA)on the expression of leptin,and apoptosis,proliferation and intracellular Ca2+concentration([Ca2+]i)of hepatic stellate cells(HSCs)and to provide evidence for liver fibrosis gene therapy.Methods HSCs were divided into normal control group,blank vector group,siRNA negative control group and leptin-siRNA group.After transfection of the leptin-siRNAs into HSCs,cell proliferation was measured by MTT assay.Cell cycle and apoptosis were measured by flow cytometry.Expression of leptin was detected by immunocytochemistry and Western blot.[Ca2+]iwas measured by Fura-2/AM loading.Results Compared with the normal control group,the blank vector group and the siRNA control group,the protein expression of leptin and the cell growth were significantly inhibited in the leptin-siRNA group(P<0.05).The proliferation rate of HSCs was significantly different at different time points(24,48 and 72h)(P<0.05).The cell apoptosis rate was increased significantly in the leptin-siRNA group(P<0.01).At the same time,Leptin-siRNA-induced[Ca2+]iwas also significantly reduced(P<0.05).Conclusion The leptin gene may play an important role in liver fibrosis progression and is potentially a novel predictive and prognostic marker for liver fibrosis.更多还原