双调控溶瘤腺病毒介导超抗原SEA基因靶向膀胱肿瘤表达

Bladder tumor targeted expression of adenovirus mediated SEA gene regulated by hTERT and PSA promoters

目的 构建携带SEA基因的选择性增殖腺病毒,体外实验观察SEA基因的表达及其刺激淋巴细胞对肿瘤的杀伤功能.方法 构建一种由端粒酶（hTERT）和缺氧反应元件（HIF）双重启动的携带SEA的选择性增殖腺病毒,通过逆转录-聚合酶链反应（RT-PCR）检测SEA在肿瘤细胞内mRNA表达,免疫荧光定位SEA表达于肿瘤细胞,Western blot测定蛋白表达,显微镜下动态观测淋巴细胞与肿瘤细胞共培养.酶联免疫吸附试验（ELISA）检测白细胞介素（IL）-4分泌.结果 琼脂糖电泳252 bp处可见清晰条带;免疫荧光及Western blot用考马斯亮蓝染色显示在27 kDa附近可见蛋白清晰表达;淋巴细胞与肿瘤细胞共培养12、24、48 h实验组肿瘤细胞明显少于对照组,ELISA检测IL-4分泌量实验组均高于对照组（t=2.585 P＜0.05）.结论 携带SEA基因的选择性腺病毒成功构建并表达目的基因,证明对肿瘤细胞的杀伤作用.

Objective SEA gene construct carrying the selective adenovirus, in vitro experimental observation SEA gene expression and to stimulate anti-tumor function of lymphocytes. Methods Building a dual boot by telomerase, and HIF-carrying SEA selective adenovirus by real-time quantitative polymerase chain reaction （PCR） detection of SEA in the nucleic acid expression in tumor cells, immune fluorescence positioned SEA expressed in tumor cells, Western blotting determination of protein expression, observed under a microscope, dynamic co-culture of lymphocytes and tumor cells, enzyme linked immu-nosorbent assay （ELISA） detection of interleukin （IL）-4 secretion. Results SEA levels of success in the nucleic acid and protein expression, cell ELISA and flow cytometry proved to be CD3＋ T lymphocytes significantly stimulated proliferation, lymphocytes and tumor cells co-cultured tumor cells 12, 24, 48 hours the experimental group significantly lower than the control group, ELISA detection of IL-4 secretion experi-mentsgroup were higher than that （t = 3.585 P 〈 0.05 ）. Conclusion The choice of carrying SEA gene adenovirus gene was successfully constructed and expressed a preliminary verification of the tumor cells in vitro.