RNAi介导MeCP2基因沉默对HSC-T6细胞活化增殖的影响  

Experimental study of proliferation and activation of HSC-T6cells through RNA inference targeting MeCP2

作　　者：陶辉[1] 黄成[1] 杨晶晶[1,2] 马陶陶[1] 张磊[1] 卞尔保[1] 李政通[1] 章慧[1] 吕雄文[1] 李俊[1] 

机构地区：[1]安徽医科大学药学院,安徽天然药物活性研究省级重点实验室,国家中医药管理局中医药三级实验室＂中药药理实验室＂,安徽合肥230032 [2]安徽医科大学第二附属医院药剂科,安徽合肥230601

出　　处：《中国药理学通报》2012年第3期333-336,共4页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 30873081,81072686)

摘　　要：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,siR-NA),通过脂质体LipofectamineTM2000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-SMAmRNA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。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 LipofectamineTM 2000.Then the corpuscular transfection efficiency was observed by inverted microscope.HSC cell proliferation was determinated by MTT.The mRNA levels of α-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 α-SMA and MeCP2 was measured by Western blot.Results MeCP2-siRNA effectively inhibits the cell proliferation,and MeCP2-siRNA significantly decreases the levels of α-SMA and MeCP2,suggesting that MeCP2 may be a potential target gene in the hepatic fibrosis.Conclusions Silencing MeCP2 expression by RNAi significantly inhibited the hepatic stellate cells proliferation,MeCP2 may be a potential therapeutic target gene for hepatic fibrosis.

关 键 词：DNA甲基化 肝星状细胞 甲基CpG结合蛋白2 细胞周期 肝纤维化 Α平滑肌肌动蛋白 

分 类 号：R-332[医药卫生] R322.47