联合自杀基因及内皮抑素基因双靶点体外治疗膀胱癌

Effects of double targeting gene therapy by suicide gene combined with endostatin gene in treat- ment of bladder cancer：an experimental study in vitro

目的观察基因重组腺相关病毒自杀基因及内皮抑素（ES）联合基因体外治疗膀胱癌的效果。方法（1）通过基因重组腺相关病毒（rAAV）-增强绿色荧光蛋白（EGFP）转染膀胱肿瘤T24细胞，确定rAAV对T24细胞的转染效率；（2）通过rAAV-胸苷激酶（TK）-核糖体插入位点（IRES）-ES（简称rAAV—TIE）体外转染T24、HUVEC细胞，MTT法、流式细胞仪等方法来检测诱导凋亡作用。结果（1）rAAV—EGFP转染T24细胞后可以表达携带的外源基因EGFP，感染效率约为62％；（2）rAAV—TK、rAAV—TIE转染T24细胞72h后，流式细胞仪检测结果显示，rAAV—TK、rAAV—TIE两组凋亡率分别是37．02％和32．14％，明显高于空病毒转染组（7．90％）和空白对照组（0．30％）。结论rAAV—TIE可有效抑制膀胱癌的血管生成和肿瘤的生长，能够双靶点基因治疗膀胱肿瘤。

Objective To investigate the effect of double targeting gene therapy by using recombinant adeno-associated virus-thymidine kinase （TK）-internal ribosome entry site （IRES）-endostatin （ES） （rAAV-TIE） in vitro. Methods （ 1 ） Bladder cancer cells of T24 were cultured and transfected by rAAV-EGFP, and the virus transfect efficacy was determined. （2） T24 and HUVEC were cultured and transfected by rAAV-ES, rAAV-MCS （blank virus） , and rAAV-TIE respectively. Seventy-two h later the levels of ES in the supernatants were measured by ELISA, and annexin V apoptosis test kit was used to examine the apoptosis. Results （ 1 ） Seventy-two h after transfection, ES could be detected in the supernatants of the T24 ceils transfected with rAAV-ES,and rAAV-TIE. （2） The apoptosis rate of the T24 cells transfected with rAAV-TK and rAAV-TIE was 34.12% and 36.91% respectively,significantly higher than that of the T24 ceils transfected with rAAV-MCS and of the control group （3.08% and 0.84% ,all P 〈 0.05）. Conclusion rAAV-TIE inhibits the tumorigenesis and angiogenesis in bladder cancer effectively. Double targeting gene therapy against bladder cancer can be achieved by using rAAV.