腺病毒介导的NT4p53(N15)Ant异源融合基因对肝癌细胞的杀伤作用

目的 构建包括神经营养因子4(NT4)信号肽,p53氨基端短活性肽(12～26位氨基酸)及蛋白质转导结构域-黑腹果蝇触足肽(Ant)序列在内的异源融合基因,并以腺病毒作为基因转移载体观察NT4p53(N15)Ant基因在体外对肝癌细胞系HepG2的杀伤效应.方法 应用互为模板的引物PCR技术及T载体克隆法获得p53(N15)Ant基因克隆,经酶切后连入pBV220/NT4质粒,再将融合基因NT4p53(N15)Ant亚克隆至腺病毒的穿梭质粒内,与辅助质粒pJM17共转染HEK293细胞,通过同源重组获得重组腺病毒,经PCR鉴定后,NT4p53(N15)Ant重组腺病毒感染HepG2细胞,用MTT比色法和PI染色流式细胞计数仪分析Ad.NT4p53(N15)Ant对肿瘤细胞和正常细胞的杀伤效应及凋亡率.结果 克隆出NT4p53(N15)Ant基因,得到高滴度的重组腺病毒,提取病毒DNA经PCR证实含目的基因.MTT测定结果显示,与平行对照病毒Ad.GFP相比,Ad.NT4p53(N15)Ant对HepG2细胞有强烈的杀伤效应,且以病毒感染后48 h作用最为明显,对肿瘤细胞抑制率达到63.3%,而对正常细胞NIH3T3的影响很小.Ad.NT4p53(N15)Ant感染HepG2细胞30 h后的流式细胞仪分析结果显示:Ad.NT4p53(N15)Ant可使HepG2细胞发生凋亡,凋亡率为18.16%.结论 通过分子克隆体外重组技术我们首次成功制备了NT4p53(N15)Ant复制缺陷型重组腺病毒;初步的研究发现Ad.NT4p53(N15)Ant对HepG2细胞有强烈的杀伤效应,这种效应部分是通过诱导HepG2细胞的凋亡而实现的.

Adenovirus-mediated killing of hepatocellular carcinoma HepG2 cells by heterogeneous fusion gene NT4p53(N15)Ant

Objective To construct a recombinant adenovirus Ad.NT4p53(N15)Ant and explore its cytotoxic effect against hepatocellular carcinoma HepG2 cells in vitro.Methods The recombinant adenovirus containing the fusion gene of neurotrophin 4(NT4)signal peptide,N-terminal residues(12-26) of p53 and 17 amino acid Drosophila homeobox protein Antennapedia(Ant) was constructed by gene cloning protocol.The effect of this fusion gene on HepG2 cells was evaluated by MTT assay,PI staining and flow cytometry.Results The fusion gene Ad.NT4p53(N15)Ant was successfully constructed,as verified by restriction endonuclease digestion and PCR.Ad.NT4p53(N15)Ant could strongly suppress the growth of HepG2 cells(with a growth inhibition rate of 63.3% 48 h after infection) without affecting NIH-3T3 cells.Flow cytometry showed that Ad.NT4p53(N15)Ant could induce obvious apoptosis of HepG2 cells.Conclusion The recombinant adenovirus containing NT4p53(N15)Ant fusion gene can inhibit the growth the of HepG2 cells in vitro partially by inducing cell apoptosis.