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构建肝细胞癌(HCC)新型靶向基因治疗体系,并以存活素(survivin)基因为靶点,探讨该体系体外杀伤肝癌细胞的作用。构建由巨细胞病毒(CMV)增强子和甲胎蛋白(AFP)启动子构成的融合启动子(AV)驱动的pcD-NA3.1(-)AV质粒,及含绿色荧光蛋白(GFP)的pcDNA3.1(-)AVGFP和含siRNA-survivin的pcDNA3.1(-)AV-siRNA-survivin质粒,以磷酸钙纳米为载体,导入HepG2、SMMC-7721和Hela细胞中,观察转染效率及体外对肝癌细胞的杀伤效应,并采用流式细胞技术(FCM)检测细胞凋亡率。结果显示:AV启动子可特异性驱动下游基因在HepG2细胞表达。此外,pcDNA3.1(-)AVsiRNA-survivin在HepG2细胞中可有效沉默survivin的mRNA和蛋白表达,并诱导68.8%的细胞死亡,而在Hela和SMMC-7721细胞中无显著作用。生长曲线结果显示,转染了pcD-NA3.1(-)AVsiRNA-survivin的HepG2细胞的生长显著受抑(P<0.05)。结果表明,该新型靶向基因治疗体系可高选择性作用于肝癌细胞,可为临床肝癌的基因治疗提供新的研究思路。
To construct a novel targeted gene therapy system for hepatocellular carcinoma (HCC) and to investigate the effect of the system on killing hepatocarcinoma cells by targeting the survivin gene. A fusion promoter (AV) driven pcD-NA3.1 (-) AV plasmid consisting of cytomegalovirus (CMV) enhancer and AFP promoter was constructed and pcDNA3 containing green fluorescent protein (GFP) .1 (-) AVGFP and pcDNA3.1 (-) AV-siRNA-survivin containing siRNA-survivin were transfected into HepG2, SMMC-7721 and Hela cells with calcium phosphate nano- Hepatocarcinoma cells were killed and the rate of apoptosis was detected by flow cytometry (FCM). The results showed that: AV promoter can specifically drive downstream genes expressed in HepG2 cells. In addition, pcDNA3.1 (-) AVsiRNA-survivin effectively silences survivin mRNA and protein expression in HepG2 cells and induces 68.8% of cell death without significant effect in Hela and SMMC-7721 cells. The growth curve showed that the growth of HepG2 cells transfected with pcD-NA3.1 (-) AVsiRNA-survivin was significantly inhibited (P <0.05). The results show that the new targeted gene therapy system can selectively act on hepatocellular carcinoma cells and provide new research ideas for the gene therapy of clinical liver cancer.