HSV－TK基因治疗系统对人胃癌细胞的杀伤作用

Gene Therapy for Human Gastric Cancer Using Thymidine Kinase Gene of Herpes Simlex Virus

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: The aim is to construct a recombinant retrovirus vector that expresses geneof herpes simplex virus thymidine kinase (HSV-TK) in eukaryotic cells and to trans feet HSVTK gene in the vector into gastric carcinoma cells and to observe retrovirus-mediated gene therapy using HSV-TK gene/GCV (ganciclovir) system for human gastric carcinoma. Metheds:HSV-TK gene(1. 7 kb) was digested with restriction enzymes EcoRI and Sail and isolated fromplasmid plC19R/MCI-TK, and then directionally inserted into retroviral vector pDOR-neo. TheHSV-TK gene packaged in the vector was trans feeted into SGC 7901 gastric carcinoma cells(SGC 7901/TK cell) by LIPOFECT AMINE. Sensitivity of SGC 7901/TK cell to GCV was examined in vitro' Moreover, SGC 7901/TK cells were innoculated into nude mice subcutaneously,and the effect of GCV therapy was examined in vivo. Results: A recombinant retrovirus vector,named PDTKZ expressing HSV-TK gene in eukaryotic cells, was successfully constructed andtransfeted into SGC 7901 cells. The SGC 7901/TK cells were sensitive to GCV in vitro in adose- and time-dependent manner, with IC,, as 2. 1μg/ml in 72h, and the toxicity was also seento the nearby parental SGC 7901 cells, so discribed as by stander effect n. Moreover, in vivostudies demonstrated that tumors containing HSV-TK gene regressed when the animals weretreated with GCV. Conclusion: Retrovirally transmitted HSV-TK gene therapy with GCV mayprovide a new therapeutic approach for treatment of gastric carcinoma.