甲胎蛋白启动子调控表达p53基因的肝癌细胞靶向性基因治疗？…

目的 利用含有人甲胎蛋白启动子的腺相关病毒载体，构建能在人肝癌细胞株中特异表达目的的基因的质粒。方法 通过设计含有特定酶切点位点的引物，选择性地从人基因组中扩增出人甲胎蛋启动子（ＡＦＰ ｐｒｏｍｏｔｅｒ）主列并克琶含有绿色荧光蛋白（ＧＦＰ报基因的质粒，ｐＲＴ－ＵＦ５上，构建成含报告基因的重组腺相关病毒质粒ｒＡＡＶ－ＡＦＰ－ＧＦＰ转染表达ＡＦＰ的Ｈｅｐ Ｇ２细胞株和不表达ＡＦＰ的２９３因的质粒ｒＡＡ

Construction of a hepatoma targeting vector of adeno associated virus containing human alpha fetoprotein promoter and wild p53 gene in gene therapy of liver cancer

Objective To construct plasmids that express target genes in hepatoma cell line using adeno associated virus (AAV)vectors containing human AFP promoter. Methods Primers containing specific enzyme cutting sites were designed to amplify the alpha fetoprotein promoter(AFP promoter) from human genome. The promoter was cloned into pTR UF5, a plasmid containing GFP reporter gene,resulting in the recombinant AAV plasmid containing the reporter gene (rAAV AFP GFP). Blunted ligation was used to construct the recombinant AAV vector plasmid containing human wild p53 gene (rAAV AFP 53). The plasmid rAAV AFP GFP was used to transfect the AFP expressing Hep G 2 and non AFP expressing 293 cell lines, respectively, to measure the function of the cloned AFP promoter. Flow cytometry was used to measure the effect of rAAV AFP 53 on hepatoma cell line HLE. Results rAAV AFP 53 and rAAV AFP GFP were verified by DNA sequencing and enzyme digestion to carry human AFP promoter. Cell transfection of rAAV AFP GFP showed selective expression in AFP positive hepatoma cell lines with a transfection rate of 36.5%; rAAV AFP 53 induced apoptosis rate was 73.88%. Conclusion Two adeno associated virus plasmids are successfully constructed that carry p53 gene and reporter gene, respectively, guided by AFP promoter. The former one showes a hepatoma specific apoptosis inducing effect.