双调控溶瘤腺病毒对膀胱肿瘤EJ细胞的杀伤作用

Anti-tumor effect of dual-regulated oncolytic adenovirus on human EJ bladder cancer cells in a in vitro model

目的:构建由人端粒酶启动子(hTERT)和缺氧诱导因子启动子(HIF)双向调控的携带SEA基因的选择性增殖腺病毒,体外试验其对肿瘤的杀伤功能。方法:将hTERT和HIF分别酶切、连接在缺陷型腺病毒的穿梭质粒的E1A和E1B序列前,调控E1A和E1B蛋白的表达,共同转染人胚肾293细胞进行同源重组,获得双调控选择性复制性腺病毒,进行PCR鉴定。获取病毒转染膀胱肿瘤细胞EJ,MTT检测细胞存活和生长情况。结果: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;MTT检测实验组在12、24、48 h活细胞数低于对照组。结论:成功构建了携带SEA基因的选择性腺病毒,且其可表达目的基因,对膀胱肿瘤细胞有一定的杀伤作用。

Objective： To construct a tumor-selective, replication competent adenovirus carrying regulated by human telomerase reverse transcriptase （hTERT） and hypoxia- inducible factor （HIF） SEA gene and promoters and investigate its anti-tumor effect in vitro. Methods： hTERT and HIF were digested, and then ligated to the shuttle plasmid of the replication-deficient adenovirus that encodes E1A/E1B sequence to regulate their were transfected into human embryonic kidney 293 cells （HEK 293 ）. The dual-regulated expressions. They , tumor- selective adenovirus was extracted through homologous recombination, and verified by PCR, so that EJ bladder cancer cells that was transfected from adenovirus could be obtained. The cell viability was measured by MTI＇ assay. Results： Through the co-culture of lymphocytes with tumor cell,the number of tumor cells in the test groups was found to be significantly smaller than that in control group at 12,24, and 48 h after treatment. Via MTT assay, it was shown that the number of viable cells in test groups was significantly smaller that in control group. Conclusion： The anti-tumorfunction of the successfully modeled/expressed adenovirus carrying SEA gene is confirmed.