转化生长因子β1及基质金属蛋白酶抑制剂1、2siRNA真核表达载体的构建与鉴定  

Construction and identification of siRNA eukaryotic expression vectors targeting on TGF β1, TIMP-1 and TIMP-2 genesin vitro

作　　者：钱克莉[1] 徐宁[2] 郎清[1] 戚敬虎[1] 孙银春[1] 肖朗[1] 刘杞[1] 石小枫[1] 

机构地区：[1]重庆医科大学附属第二医院感染科,重庆医科大学病毒性肝炎研究所教育部感染性疾病分子生物学重点实验室400010 [2]陕西省榆林市第二医院重症医学科

出　　处：《中华肝脏病杂志》2011年第4期291-296,共6页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30671867）

摘　　要：目的 用RNA干扰技术,分别以转化生长因子（TGF）β 1、基质金属蛋白酶抑制剂（TIMP）-1和TIMP-2为靶基因,设计并构建针对TGF β 1、TIMP-1和TIMP-2基因的小干扰RNA（siRNA）真核表达载体,并在体外检测其对大鼠肝星状细胞株（HSC-T6）的TGF β 1、TIMP-1和TIMP-2基因表达的抑制情况.方法 设计合成TGF β 1、TIMP-1和TIMP-2的siRNA并与含绿色荧光蛋白的pGenesil-1载体连接,构建siRNA真核表达载体,并测序鉴定.体外转染HSC-T6细胞,观察转染效率,并用荧光定量PCR以及Western blot分析对目的 基因的抑制效率.组间比较用方差分析,两两比较用q检验.结果 成功构建了针对TGF β 1、TIMP-1和TIMP-2基因的siRNA真核表达载体.体外成功转染HSC-T6细胞,转染后的细胞TGF β 1、TIMP-1及TIMP-2 mRNA表达分别下调63.4%±8.0%,64.5%±9.0%,55.0%±17.0%（F值分别为17.55、128.42、210.36,P值均＜0.01）,TGF β 1、TIMP-1及TIMP-2蛋白表达分别下降57.8%±3.0%,55.1%±5.0%,49.3%±1.0%（F值分别为130.75、159.09、35.72,P值均＜0.01）.结论 成功构建了针对TGF β 1、TIMP-1和TIMP-2基因的siRNA真核表达载体;将重组载体成功转染入体外培养的HSC-T6细胞,并显著抑制了目的 基因的表达;为进一步研究其在体抑制表达提供了实验工具.Objective To construct the siRNA eukaryotic expression vectors targeting on TGF β1,TIMP-1 and TIMP-2 and to investigate the inhibitory efficiency of target genes expression on rat hepatic stellate cell in vitro. Methods The siRNA cDNA sequences ofTGF β 1, TIMP-1 and TIMP-2 were designed,synthesized and inserted into plasmid pGenesil-1 respectively to generate eukaryotic expression plasmids.The plasmids were transfected into HSC T6 cells in vitro and the inhibitory efficiency of target genes expression was observed with real-time PCR and Western blot. Results The eukaryotic expression vectors were constructed successfully. The expressions of TGF β1 mRNA, TIMP-1 mRNA and TIMP-2mRNA in siRNAtransfected groups were decreased by 63.4% ± 8.0%, 64.5% ± 9.0% and 55.0% ± 17.0% respectively and the expressions of TGF β1 protein, TIMP-1 protein and TIMP-2 protein were decreased by 57.8% ± 3.0%,55.1% ± 5.0%, 49.3% ± 1.0% respectively as compared to the control groups. Conclusions The siRNA eukaryotic expression vectors constructed targeting on TGF β1, TIMP-1 and TIMP-2 could reduce the expressions of target genes and they might be able to used for the exploration of new anti-fibrosis drugs genetically.

关 键 词：肝硬化 转化生长因子Β 肝星状细胞 

分 类 号：R285.5[医药卫生—中药学]