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目的：构建单纯疱疹病毒脱氧胸苷激酶（ＨＳＶ－ＴＫ）基因的重组逆转录病毒真核表达载体，并将其转染胃癌细胞，观察ＨＳＶ－ＴＫ／ＧＣＶ（丙氧鸟苷）基因治疗系统对胃癌细胞的杀伤作用。方法：用ＥｃｏＲＩ和ＳａｌＩ酶切质粒ｐＩＣ１９Ｒ／ＭＣＩ－ＴＫ，回收１．７ｋｂ的ＨＳＶ－ＴＫ基因片段，将其定向克隆入逆转录病毒载体ｐＤＯＲ－ｎｅｏ的多克隆位点。用Ｌ（ＩＰＯＦＥＣＴ）ＡＭＩＮＥ法将ＨＳＶ－ＴＫ基因转染胃癌细胞系ＳＧＣ７９０１。测定阳性转染细胞（ＳＧＣ７９０ＶＴＫ细胞）在体外对ＧＣＶ的敏感性，并将其接种于裸鼠皮下，观察ＧＣＶ在体内对胃癌细胞的抑制作用。结果：经酶切鉴定，成功地构建了ＨＳＶ－ＴＫ基因的真核表达载体ｐＤＴＫＺ，并将其转入胃癌细胞。体外实验表明，ＧＣＶ对ＳＧＣ７９０１／ＴＫ细胞有明显的杀伤作用，其作用呈现剂量和时间依赖性特点，且同时表现出对其周围亲代ＳＧＣ７９０１细胞的杀伤效应（旁观者效应）。体内实验表明，ＧＣＶ可抑制ＨＳＶ－ＴＫ阳性胃癌细胞的生长，使已形成的肿瘤逐渐消失。而ＧＣＶ对ＨＳＶ－ＴＫ阴性的亲代ＳＧＣ７９０１细胞在体内外均无明显的杀伤和抑瘤作用。结论：结果表明，逆转录病毒载体介导的ＨＳＶ－ＴＫ基因转移胃?
Objective: To construct a recombinant retrovirus eukaryotic expression vector for herpes simplex virus deoxythymidine kinase (HSV-TK) gene and transfect it into gastric cancer cells to observe the HSV-TK/GCV (glycosin) gene therapy system The killing effect of gastric cancer cells. METHODS: Plasmid pIC19R/MCI-TK was digested with EcoRI and SalI and the 1.7 kb HSV-TK gene fragment was recovered and cloned into the multiple cloning site of retroviral vector pDOR-neo. The HSV-TK gene was transfected into gastric cancer cell line SGC7901 by L (IPOFECT) AMINE method. The sensitivity of positively transfected cells (SGC790VTK cells) to GCV in vitro was measured and inoculated subcutaneously in nude mice to observe the inhibitory effect of GCV on gastric cancer cells in vivo. RESULTS: After enzyme digestion, the eukaryotic expression vector pDTKZ of HSV-TK gene was successfully constructed and transferred into gastric cancer cells. In vitro experiments showed that GCV had a significant killing effect on SGC7901/TK cells, and its effect was dose- and time-dependent, and at the same time showed a killing effect (bystander effect) on its surrounding parental SGC7901 cells. In vivo experiments showed that GCV can inhibit the growth of HSV-TK positive gastric cancer cells, and the formed tumors gradually disappeared. GCV had no significant killing and antitumor effects on HSV-TK-negative parental SGC7901 cells in vitro and in vivo. Conclusions: The results indicate that retroviral vectors mediate HSV-TK gene transfer to the stomach?