应用重组分泌型内皮抑素腺相关病毒治疗膀胱癌的实验研究

Gene therapy of bladder cancer by using recombinant adeno-associated virus-endostatin:experiments in vitro and in vivo L

目的包装重组分泌型内皮抑素腺相关病毒(rAAV-ES)并研究其在体内外的抗肿瘤作用。方法采用质粒共转染方法包装 rAAV-ES;转染(感染复数 MOI=10~5)膀胱癌细胞(EJ)后,ELISA 法测定上清液中重组内皮抑素的浓度并检测其对血管内皮细胞趋化运动的抑制作用;建立裸鼠肿瘤模型,检测 EJ 细胞被转染后的成瘤率及全身应用 rAAV-ES 后抑制肿瘤发展的作用及其毒副作用。结果成功包装具有生物活性的 rAAV-ES,病毒滴度为1.0×10^(12)v·g/ml;转染 EJ 细胞后上清液中重组内皮抑素浓度为54.09 ng/ml,对血管内皮细胞趋化运动的抑制率为37.45%;被 rAAV-ES转染后 EJ 细胞的成瘤率仅为对照组的2/5,体内实验证实全身应用 rAAV-ES 后血清中内皮抑素长期高效表达(30～40 ng/ml),肿瘤生长速度慢(32 d±9 d),瘤体微血管密度低[(8±3)条/0.739 mm^2],与对照组比较,差异有统计学意义(均 P<0.05),心脑组织学检查未见缺血和其他异常改变。结论rAAV-ES 无毒副作用,可有效地抑制肿瘤的血管生成,从而抑制膀胱癌的发生、发展,其成功包装为原位基因治疗膀胱癌奠定了基础。

Objective To package recombinant adeno-associated virus-endostatin （rAAV-ES） and study its anti-tumor effect in vitro and in vivo. Methods rAAV-ES was packaged with co-transfection technique and transfected into the human bladder cancer cells of the line EJ. 24 h later ELISA was used to examine the concentration of ES in the supernatant. The inhibition of human umbilical veins endothelial cells （HUVECs） chemotactic movement were examined by Transwell system. Nude Balb/c mice were divided into 4 groups： （ 1 ） 5 mice were inoculated with the EJ cells transfected with rAAV-ES or rAAV-enhanced yellow fluororescence protein （rAAV-EYFP） for 3 days to the subcutaneous tissues of bilateral shoulders so as to observe the growth of tumor. （2） 24 mice were injected with rAAV-ES intramuscularly and then the serum ES was examined every 10 days since the 10 th day after the injection. （3） 36 mice were randomly subdivided into 3 equal subgroups to be injected with rAAV-ES, rAAV-EYFP, or RPMI medium, inoculated with EJ cells 2 weeks later, and then killed 50 days later to observe the size of tumor. （4） 4 healthy mice and 4 mice injected with rAAV-ES for 8 weeks were killed with their hearts and brains taken out to observe the side effects. Results rAAV-ES was packaged successfully. The ES concentration in the supernatant of culture fluid of the EJ cell transfected with rAAV-ES was 54.09 ng/ml. The inhibition rate of the HUVECs chemotactic movement was 37.45%. The xenograft formation rate was 2/5 for the EJ cells transfected with rAAV-ES. The serum ES levels of the mice injected with rAAV-ES remained high. The tumor size in the mice injected with rAAV-ES was significantly smaller than those of the other groups （ both P 〈 0.01 ）. No pathological changes was found in the hearts and brains in the mice injected with rAAV-ES. Conclusion rAAV-ES inhibits tumor angiogenesis, and tumor formation and progression. Successful packaging of rAAVES has laid a foundation for gene therapy of bladder cancer.