基因重组腺相关病毒内皮抑素基因治疗膀胱癌的实验研究

Effects of gene therapy by rAAV-Endostatin in treatment of bladder cancer： an experimental study

目的探讨基因重组腺相关病毒（rAAV）内皮抑素（ES）基因治疗膀胱癌的效果。方法通过rAAV增强绿色荧光蛋白（EGFP）转染膀胱肿瘤T24细胞株确定rAAV对细胞的转染效率；rAAV—ES体外转染T24细胞及人脐静脉血管内皮细胞（HUVEC），偶氮唑盐法、流式细胞仪检测其对T24细胞及HUVEC细胞凋亡的诱导作用；构建裸鼠膀胱癌模型，观察肿瘤生长情况及测量微血管密度（MVD），分析rAAVES基因治疗膀胱癌的体内效果。结果rAAV—EGFP转染T24细胞后可以稳定表达EGFP；rAAV—ES转染T24细胞72h后，细胞凋亡率37．02％，明显高于空病毒转染组（7．90％）和空白对照组（0．30％），差异均有统计学意义（均P〈0．05）。荷瘤裸鼠瘤内注射rAAV—ES9d后，肿瘤生长受到明显抑制；治疗4周结束后rAAV-ES组、rAAV-MCS组和空白对照组肿瘤体积分别为（0．75±0．08）、（1．27±0．13）、（1．24±0．17）cm^3，差异有统计学意义（P〈0．05）。结论体内外实验表明rAAV—ES可有效抑制膀胱癌的血管生成和肿瘤生长。

Objective To investigate the effect of gene therapy by using recombinant adeno-associated virus endostatin （ES） （rAAV-ES） for bladder cancer. Methods Bladder cancer cell line T24 was cultured and transfeeted with rAAV-ES and rAAV-MCS （vehicle control）. 72 hours after transfection, the level of ES in the supernatants was measured by ELISA and Annexin Ⅴ apoptosis test kit was used to examine the apoptosis. Human umbilical vein endothelial cells （HUVECs） were transfected with rAAV-ES and rAAV-MCS. MTT method and flow cytometry were used to detect the apoptosis of the HUVECs. Balb/c nude mice were inoculated subcutaneously with T24 cells. Fifteen mice with tumor were randomly divided into 3 groups to be treated by rAAV-MCS and rAAV ES, and 5 mice were used as control group. Four weeks after treatment, blood samples were collected to detect the ES level by ELISA. The tumors were taken out for microscopy to calculate the microvessel density （MVD）. Results Seventy-two h after transfeetion, ES could be detected in the supernatants of the T24 cells transfected with rAAV-ES. The apoptotic rates of the T24 cells transfected with rAAV-ES was 37.02%, significantly higher than those of the T24 cells transfected with rAAV-MCS and of the control group （P〈0. 05）. Transfection of rAAV ES increased the apoptotic rate of the HUVECs time-dependently. Four weeks after the transfection, the tumor volumes of the rAAV-ES group was （0.75±0.08）cm^3 , significantly lower than those of the rAAV-MCS（1.27±0.13）cm3 group and con-trol group（1. 24±0.17）cma （P〈0.05）. Conclusions rAAV-ES could effectively inhibits the tumor growth and angiogenesis in bladder cancer.