胸苷激酶基因治疗胃癌的体外实验

The Experiment of HSV-tk Gene Therapy for Gastric Carcinoma in vitro

将单纯疱疹病毒胸苷激酶基因（ＨＳＶ－ｔｋ）导入恶性肿瘤细胞，随后可应用药物丙氧鸟苷（ｇａｎｃｉｃｌｏｖｉｒ， ＧＣＶ）选择性杀死肿瘤细胞．构建了含胸苷激酶与潮霉素磷酸转移酶（ｈｐｈ）融和基因（ＨｙｔＫ）的真核表达载体ＬＸｐｓｐ－ＨｙｔＫ．以脂质体（ｌｉｐｏｆｅｃｔｉｎ）为介导，将这种质粒与仅含潮霉素Ｂ基因的质粒ＬＸＳＨ 分别转染胃癌细胞系ＢＧＣ－８２３，用６０ Ｕ／ｍ ｌ潮霉素Ｂ进行筛选，得到了可稳定传代的阳性克隆，分别命名为ＢＧＣ－ＨｙｔＫ 和ＢＧＣ－Ｈｙ．三种细胞的生长曲线无明显差别．用不同浓度的ＧＣＶ 分别作用于ＢＧＣ－ＨｙｔＫ， ＢＧＣ－Ｈｙ 及ＢＧＣ－８２３，０．０２～２００ μｇ／ｍ ｌ 的ＧＣＶ 对ＢＧＣ－ＨｙｔＫ 细胞有明显的杀伤作用（ＩＣ５０＝ ０．０２ μｇ／ｍ ｌ），而对另外两种细胞几乎无毒性作用（ＩＣ５０＞ ２００μｇ／ｍ ｌ）．２０ μｇ／ｍ ｌＧＣＶ 作用９６ ｈ 后，仅存在２０％ 的ＢＧＣ－ＨｙｔＫ 就可使周围的大部分ＨＳＶ－ｔｋ－ 的肿瘤细胞死亡，说明存在较显著的“旁观者效应”

Transfer of the herpes simplex virus thymidine kinase (HSV tk) gene into malignant tumor cells could confer the tumor cells susceptibility to antiviral drug ganciclovir (GCV), and then kill the tumor cells. A vector LXpsp HytK which contains the fusion gene (HytK) of HSV tk gene and hygromycin phosphotransferase gene (hph) was constructed. This vector and plasmid LXSH which only contained hph were introduced into a gastric carcinoma cell line BGC 823 respectively by lipofectin. Hytk + and Hy + cell clones were obtained under the pressure of 60 U/ml hygromycin B and named BGC HytK and BGC Hy. There was no difference in the growth curve of these three kinds of cells. The cytotoxic effect on BGC HytK cells exposed to GCV ( 0.02 ～200 μg/ml) was obvious ( IC 50 =0 02 μg/ml),while GCV almost had not any cytotoxic effect on BGC 823 and BGC Hy cells ( IC 50 >200 μg/ml). Exposed to 20 μg/ml GCV for 96 hours, most of the cells were killed when BGC HytK occupied only 20% of the total culture cells, showing obvious bystander effect.