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胃癌是威胁人类最常见的恶性肿瘤之一,目前的治疗方法主要是手术和化疗.但对中晚期胃癌的疗效却欠理想.基因治疗为恶性肿瘤的治疗开辟了新的途径.自杀基因(suicide gene)为目前研究较多的基因.TK 基因(单纯疱疹病毒胸苷激酶基因 herpes simplx virus-thymidine kinase,HSV-TK)即为自杀基因之一,它能将其作用的前药9-丙氧鸟苷(ganciclovir,GCV)转化为磷酸化.GCV,干扰细胞 DNA 合成,导致转基因细胞的死亡.但自杀基因抗肿瘤更重要的作用是通过产生旁观者效应(bystander effect),所谓旁观者效应就是不仅转基因细胞被杀死,同时伴有周围大量未转基因的细胞亦被杀死.我们构建 TK 基因逆转录病毒表达载体,体外转染胃癌细胞株,检测其体外杀伤性.并通过转染小鼠模型胃癌,研究其体内抗肿瘤作用.1材料和方法1.1材料 TK 基因通过酶切位点 EcoRⅠ和 BamHⅠ构建于逆转录病毒载体 pLxSN.应用 PA317细胞包装逆转录病毒,培养液为 DMEM.NIH3T3为小鼠成纤维细胞,用作病毒滴度的测定.MFC 细胞为615小鼠前胃癌细胞,培养基为 DMEM,含小牛血清,置37℃培养箱培养.应用脂质体方法(按说明)将逆转录病毒质粒转染 PA317细胞,并用 G418筛选,收集含病毒上清.用 NIH3T3细胞测定病毒滴度.病毒上清经测定滴度,最高为1×10~8cfu·L~(-1).
Gastric cancer is one of the most common malignant tumors that threaten human beings. At present, the main treatment methods are surgery and chemotherapy, but the effect is not ideal for the treatment of advanced gastric cancer.Geneotherapy opens up a new way for the treatment of malignant tumors.suicide gene) is one of the most studied genes in recent years.TK gene (herpes simplex virus-thymidine kinase, HSV-TK) is one of the suicide genes, Guanyuan (ganciclovir, GCV) into phosphorylation.GCV, interfere with cell DNA synthesis, leading to the death of transgenic cells.But suicide genes more important role in anti-tumor through the bystander effect (bystander effect), the so-called bystander effect is Not only the transgenic cells were killed, but also a large number of non-transgenic surrounding cells were also killed.We constructed the TK gene retroviral expression vector and in vitro transfected gastric cancer cell lines to test its in vitro cytotoxicity and by transfection of mice Model of gastric cancer to study its antitumor effect in vivo.1 Materials and methods 1.1 Materials TK gene was constructed by enzyme digestion sites EcoR Ⅰ and BamH Ⅰ retroviral vector pLxSN.Application of P A317 cell packaging retrovirus, the culture medium for DMEM.NIH3T3 mouse fibroblasts, used as a measure of virus titer.MFC cells for 615 mouse gastric cancer cells, medium DMEM, containing fetal calf serum, set 37 ℃ incubator.Using liposome method (according to the instructions) will be retroviral plasmid was transfected into PA317 cells and screened with G418, the virus-containing supernatant was collected with NIH3T3 cells to measure the virus titer.Viral supernatant was determined by titer, The maximum is 1 × 10 ~ 8cfu · L -1.