逆转录病毒介导HyTK 基因治疗膀胱癌的实验研究

Retroviral mediated transfer of a hygromycin phosphotransferase thymidine kinase fusion gene into human bladder carcinoma cell

目的 了解逆转录病毒介导的潮霉素磷酸转移酶和 Ｈ Ｓ Ｖ Ｔ Ｋ 的融合基因（ Ｈｙ Ｔ Ｋ） 的基因转移联合更昔洛韦（ Ｇ Ｃ Ｖ） 对膀胱癌细胞的杀伤作用。 方法 构建重组质粒ｐ Ｌ（ Ｈｙ Ｔ Ｋ） Ｓ Ｎ，应用非脂质体基因转导系统 Ｆｕ Ｇ Ｅ Ｎ Ｅ Ｔ Ｍ６ 及“乒乓效应”（ｐｉｎｇｐｏｎｇ ｍｅｃｈａｎｉｓｍ） ，获得高滴度重组病毒并转染膀胱癌细胞株 Ｅ Ｊ，增强型绿色荧光蛋白（ Ｅ Ｇ Ｆ Ｐ） 报告基因检测转移效率，检测 Ｈｙ Ｔ Ｋ 基因的表达及 Ｇ Ｃ Ｖ 对转基因细胞 Ｅ Ｊ／ Ｈｙ Ｔ Ｋ 的杀伤作用。 结果 本实验逆转录病毒的转染效率为３５ ％ 左右， Ｓｏｕｔｈｅｒｎ Ｂｌｏｔ、 Ｄｏｔ Ｂｌｏｔ 及免疫组化检测证实 Ｈｙ Ｔ Ｋ 在转基因细胞的表达， Ｍ Ｔ Ｔ法测定示 Ｇ Ｃ Ｖ 对 Ｅ Ｊ／ Ｈｙ Ｔ Ｋ 细胞有明显的杀伤作用。其 Ｉ Ｃ５０ 值为１０７μｇ／ｍｌ，而对其亲代 Ｅ Ｊ细胞无明显杀伤作用（ Ｐ＜ ００５） 。按不同比例混合 Ｅ Ｊ／ Ｈｙ Ｔ Ｋ 细胞和亲代 Ｅ Ｊ细胞，发现 Ｇ Ｃ Ｖ 不仅能杀伤转入 Ｔ Ｋ 基因的细胞，而且对其周围未转基因的膀胱癌细胞存在旁观者效应，此效应并无细胞株间的特异性。 结论 逆转录病毒介导的 Ｈｙ Ｔ Ｋ

Objective To evaluate the therapeutic efficacy of retroviral mediated hygromycin phosphotransferase thymidine kinase fusion gene (HyTK)/GCV on human bladder carcinoma cell. Methods A retroviral expression vector pL(HyTK)SN was constructed.By using FuGENE TM 6 mediated transfection and “ping pong effect”technique, high titer of retroviral supernatant was obtained and HyTK gene was transferred into EJ cells. A retroviral vector encoding, enhanced green fluorescent protein,EGFP was used to rapidly detect the transduction efficiency. Antitumor effects were observed after GCV treatment. Results In vitro experiments demonstrated the EJ cells transferred by HyTK gene were killed in the GCV treatment. Nontransduced parental cells were not sensitive to GCV, but they were dead by the bystander killing of neighboring cells when mixed with EJ/HyTK cells at various ratios. In addition, this not only affect wild type EJ cells but also cells from different bladder carcinoma cell lines. Conclusions Retroviral mediated HyTK/GCV systems were a promising suicide gene therapy for bladder carcinoma. EGFP may act as a convenient and rapid reporter to monitor retroviral mediated gene transfer and expression in bladder carcinoma cells.