COL1A1-shRNA表达质粒构建及抑制COL1A1表达的有效序列的筛选

Construction of COL1A1shRNA expression plasmid and screening of effective sequences to inhibit COL1A1 expression

目的:构建和筛选对大鼠肝星状细胞前Ⅰ型胶原α1链(COL1A1)mRNA有抑制作用的COL1A1短发夹RNA(shRNA)的表达质粒.方法:从NCBI网站获得大鼠的COL1A1cDNA序列,根据Whitehead研究所的siRNA设计软件设计3条理论上最佳的siRNA序列,相应的双链DNA被插入pGPU6/GFP/Neo质粒中,即pGPU6/GFP/Neo-shRNA-A、pGPU6/GFP/Neo-shRNA-B和pGPU6/GFP/Neo-shRNA-C.为得到高效沉默COL1A1-siRNA,以脂质体LipofectAMINE2000,将1、2、3、4μgDNA质粒转染至HSC-T6细胞中,并观察转染效果.将最佳沉默siRNA导入HSC-T6细胞,RT-PCR分析各组的COL1A1mRNA表达水平.结果:靶向COL1A1mRNA的3个shRNA重组质粒载体pGPU6/GFP/Neo-shRNA-A、pGPU6/GFP/Neo-shRNA-B和pGPU6/GFP/Neo-shRNA-C经测序分析,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功.1、2、3、4μg组转染效率分别为16.7%、20.3%、23.5%和22.3%,以2μgsiRNA为最佳剂量,pGPU6/GFP/Neo-shRNA-A、pGPU6/GFP/Neo-shRNA-B和pGPU6/GFP/Neo-shRNA-C对COL1A1 mRNA的抑制率分别为16.6%,63.3%和80.3%.结论:筛选出的pGPU6/GFP/Neo-shRNA-C表达质粒能高效地抑制转染细胞COL1A1mRNA的表达,从而为肝纤维治疗提供新的方法和材料.

AIM： To construct and select procollagen type 1 alpha 1 （COL1A1） short hairpin RNA （shRNA） expression plasmid that can inhibit COL1A1 mRNA expression in rat hepatic stellate cell （HSC）. METHODS： Rat COL1A1 cDNA sequence was obtained from NCBI website. Three small interfering RNA sequences were selected through online design of the Whitehead Institute. The corresponding double-stranded DNA was used to construct pGPU6/GFP/Neo plasmids, namely pGPU6/GFP/Neo-shRNA-A, pGPU6/GFP/Neo-shRNA-B and pGPU6/GFP/Neo- shRNA-C. HSC-T6 cells were transfected with a green fluorescent protein （GFP）-labeled siRNA to assess the transfection efficiency. To get most effective and optimal dosage siRNA, the three plasmids （1, 2, 3, 4 μg） were transfected into HSC-T6 cells with Lipofectamine 2000 respectively, and the untreated HSC-T6 cells were used as controls. The expression of COL1A1 mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR） after the most effective and optimal dosage was used. RESULTS： The expression plasmids targeting on COL1A1 mRNA were successfully constructed, and confirmed by agarose electrophoresis and sequence analysis. The transfection efficiencies at a dose of 1, 2, 3, and 4 μg were approximately 16.7%, 20.3%, 23.5%, and 22.3%, and 2μg was considered as the most optimal dosage in each group. The inhibitory rates of COL1A1 mRNA levels in the HSC-T6 cells transfected with pGPU6/GFP/Neo-shRNA-A, pGPU6/ GFP/Neo-shRNA-B, and pGPU6/GFP/Neo- shRNA-C were 16.6%, 63.3%, and 80.3%, respectively, when 2 μg siRNA plasmid was used. CONCLUSION： The constructed expression plasmid pGPU6/GFP/Neo-shRNA-C can effectively inhibit the expression of COL1A1 mRNA, providing a new method and material for the treatment of liver fibrosis.