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[目的]构建同时干扰大鼠结缔组织生长因子(CTGF)2个不同基因位点的shRNA质粒,并将其转染肝星状细胞(HSC),研究其对CTGF基因表达的影响。[方法]针对大鼠CTGF mRNA靶向序列设计并合成2对DNA模版序列,用PCR的方法将2条CTGF靶向特异性的shRNA分别链接到质粒pGenesil1.2载体的U6启动子和H1启动子。将构建的干扰质粒以阳离子脂质体法转染HSC-T6细胞,应用RT-PCR及Western Blot检测CTGF的表达。[结果]成功构建CTGF shRNA重组质粒;通过RT-PCR和Western Blot分析发现干扰质粒组的CTGF mRNA与蛋白表达水平明显下降(P<0.05)。[结论]重组CTGF shRNA能有效抑制HSC中CTGF的表达。
[Objective] The purpose of this study was to construct shRNA plasmids that simultaneously interfere with two different loci of connective tissue growth factor (CTGF) in rats and transfect them into hepatic stellate cells (HSCs) to study their effect on CTGF gene expression. [Method] According to the target sequence of rat CTGF mRNA, two pairs of DNA template sequences were designed and synthesized. Two CTGF targeting specific shRNAs were respectively linked to U6 promoter and H1 promoter of plasmid pGenesil1.2 vector by PCR . The constructed interference plasmids were transfected into HSC-T6 cells by cationic liposome method, and the expression of CTGF was detected by RT-PCR and Western Blot. [Result] CTGF shRNA recombinant plasmid was successfully constructed. The expression of CTGF mRNA and protein in interfering plasmid group was significantly decreased by RT-PCR and Western Blot (P <0.05). [Conclusion] Recombinant CTGF shRNA can effectively inhibit the expression of CTGF in HSC.