RNAi介导MeCP2基因沉默对HSC-T6细胞活化增殖的影响

摘要

Aim To investigate the effect of cell proliferation in HSC-T6 cells by RNA interference inhibiting expression of MeCP2 gene. Methods siRNA targeting MeCP2 was designed and synthesized according to its mRNA, and their corresponding expression vectors were constructed and transfected into HSC-T6 cells with Li-pofectamine? 2000. Then the corpuscular transfection efficiency was observed by inverted microscope. HSC cell proliferation was determinated by MTT. The mRNA levels of a-SMA, MeCP2 in the supernatant of the cell culture were measured by Quantitative Real-Time PCR. HSC cell cycle was detected by flow cytometry of DNA content in each phase. The protein level of a-SMA and MeCP2 was measured by Western blot. Results MeCP2-siRNA effectively inhibits the cell proliferation, and MeCP2-siRNA significantly decreases the levels of a-SMA and MeCP2, suggesting that MeCP2 may be a potential target gene in the hepatic fi-brosis. Conclusions Silencing MeCP2 expression by RNAi significantly inhibited the hepatic stellate cells proliferation, MeCP2 may be a potential therapeutic target gene for hepatic fibrosis.%目的 探讨RNA干扰介导甲基CpG结合蛋白2(methyl-CpG-binding protein 2,MeCP2)基因沉默对大鼠肝星状细胞HSC-T6细胞增殖的影响.方法根据MeCP2的碱基序列设计并合成小干扰RNA(small interfering RNA,siRNA),通过脂质体LipofectamineTM 2000转染到HSC-T6细胞内,荧光倒置显微镜观察细胞的转染效率,用四甲基偶氮唑盐(MTT)法检测HSC-T6细胞增殖变化;Quantitative Real-Time PCR检测α-SMA、MeCP2 mRNA的表达;流式细胞术检测HSC-T6细胞周期的变化;Western blot检测α-SMA、MeCP2蛋白的表达.结果将MeCP2-siRNA转染进HSC-T6细胞内,MeCP2基因及蛋白水平明显降低;同时α-SMA mRNA及α-SMA 蛋白的表达水平亦明显降低;靶向封闭MeCP2基因的表达可明显抑制HSC-T6细胞的活化增殖.结论 靶向封闭MeCP2的表达可明显抑制HSC-T6细胞活化增殖,MeCP2可能是潜在的肝纤维化治疗靶点.