RNAi抑制肝星状细胞TIMP-1基因表达及对细胞生物学行为的影响  

RNAi Inhibit the Expression of TIMP-1 in CFSC and its Effect on Cell Biological Behaviour

作　　者：徐华锋[1] 邹海峰[1] 林平[1] 刘鑫[1] 刘秀萍[1] 于晓光[1] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室

出　　处：《生命科学研究》2007年第4期342-347,共6页Life Science Research

基　　金：黑龙江省卫生厅资助项目(2006-259);黑龙江省博士后启动基金(2005)

摘　　要：研究了siRNA对大鼠肝星状细胞(CFSC)TIMP-1基因表达及对细胞生物学行为的影响.用RT-PCR方法获得带有T7启动子的TIMP-1全基因序列,体外转录法获得TIMP-1dsRNA,Dicer酶切后得到siRNA,用质脂体将siRNA转染至CFSC,RT-PCR检测TIMP-1 mRNA的表达,MTT方法检测细胞生长,流式细胞仪检测细胞凋亡.结果成功获得TIMP-1 siRNA,将TIMP-1 siRNA转染至CFSC12、24、48h后,与对照相比,TIMP-1 mRNA的表达逐渐下降,经Quanity One(BIO-RAD,USA)分析后,其抑制率分别为49.18%、58.72%和64.73%;siRNA转染CFSC细胞后,CFSC生长受到抑制,细胞凋亡不明显.实验结果表明体外转录法得到的siRNA能有效地降低大鼠CFSC中TIMP-1的表达,明显抑制CFSC的增殖,但不直接引起CFSC的凋亡.To study the effect of the siRNA （small interference RNA） to the expression of TIMP-1 in CFSC （CCL4-cirrhotic fat-storing cells） and the cell biological behaviour, the TIMP-1 gene sequence with T7 promoter was obtained by RT-PCR. The TIMP-1 dsRNA was obtained by the method of in vitro transcription. The siRNA was obtained by Dicer. The siRNA was transfected to CFSC by liposome, and the expression of TIMP-1 was analyzed by RT-PCR. Cell growth was shown by methyl thiazolium tetrazolium （MTT）. The apoptosis of CFSC was detected by Flow Cytometry. The siRNA of TIMP-1 was obtained successfully. The resuh of RT-PCR showed the expression of TIMP-1 mRNA was inhibited gradually, which CFSC was transfected by siRNA 12, 24, 48 h respectively. Results analyzed by Quanity One showed the inhibitory rates of TIMP-1 mRNA was 49.18%, 58.72% and 64.73%. The growth of CFSC was inhibited, after transfecting siRNA to CFSC, but the apoptosis of CFSC was not evident. The siRNA in vitro transcription can reduce the level TIMP-1 in CFSC efficiently, and can inhibit the growth of CFSC, but the decreasing of TIMP-1 can not cause the apoptosis of CFSC.

关 键 词：RNAI 基质金属蛋白酶组织抑制因子-1 肝星状细胞 细胞凋亡 

分 类 号：Q492.6[生物学—生理学]