CTGF特异性siRNA慢病毒表达载体的构建及鉴定

目的:构建CTGF特异性siRNA慢病毒表达载体并观察其介导的RNA干扰(RNA interference,RNAi)对人肝癌细胞CTGF(connective tissue growth factor,CTGF)表达的影响?方法:针对已经筛选确定的人CTGF基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与Plk0.1-GFP-SP6载体连接产生shRNA 慢病毒载体,经PCR筛选阳性克隆进行DNA测序鉴定?用脂质体转染法将质粒共转染293T 细胞,将包装产生的慢病毒颗粒感染HepG2细胞,Real-time PCR?Western-blot检测CTGF mRNA和蛋白的表达?结果:PCR与DNA测序证实合成的含CTGF shRNA慢病毒载体寡核苷酸链插入正确?经RNA干扰后的靶细胞CTGF mRNA以及蛋白表达水平明显降低?结论:成功构建人CTGF基因RNA干扰慢病毒载体,体外感染HepG2细胞可有效降低CTGF mRNA和蛋白的表达,为以CTGF基因为靶点的肝癌基因治疗研究奠定了基础?

Construction and identification of lentiviral vector of siRNA specific for CTGF

Objective:To observe the influence of lentiviral vector-mediated RNA interference on expression of CTGF(connective tissue growth factor,CTGF)gene in human hepatocellular carcinoma cell line. Methods:The effective RNA interference sequence targeting CTGF gene was screened and determined according to our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was desiged,synthesized and cloned into Plk0.1-GFP-SP6 vector to construct a lentiviral vector which expressed CTGF shRNA,subsequently confirmed by PCR and DNA sequencing analysis. 293T cells were co-transfected with the plasmids using liposome transfection methods,and packaged to produce lentiviral particles. The recombinant lentiviruses were transfected into HepG2 cells,and the CTGF mRNA and protein expression were examined by Real-time PCR and Western-blot. The results were compared with those of the non-transfected and blank vector transfected HepG2 cells. Results:PCR analysis and DNA sequencing confirmed that the CTGF shRNA sequences were successfully inserted into the lentiviral vectors. After transfection with CTGF shRNA,the CTGF expression in HepG2 cells was significantly inhibited at both mRNA and protein levels compared with that in non-transfected and empty vector transfected HepG2 cells. Conclusion:Two lentiviral RNAi vectors targeting CTGF gene have been successfully constructed,and can effectively inhibit the expression of CTGF gene in HepG2 cells. It paves a way for CTGF-targeted gene therapy of human hepatocellular carcinoma.