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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 secreted endostatin Adeno-associated virus (rAAV-ES) and study its anti-tumor effects both in vitro and in vivo. Methods The rAAV-ES was packaged by plasmid co-transfection method. After transfection (MOI = 10 ~ 5) in bladder cancer cells (EJ), the concentration of recombinant endostatin in the supernatant was measured by ELISA and its effect on vascular endothelial To study the inhibitory effect of rAAV-ES on the tumorigenesis and its toxicities and side effects after establishing the model of tumor in nude mice. Results The biologically active rAAV-ES was successfully packaged and the virus titer was 1.0 × 10 ~ (12) v · g / ml. The supernatant of transfected EJ cells had a recombinant endostatin concentration of 54.09 ng / ml, The inhibitory rate of endothelial cell chemotactic activity was 37.45%. The tumorigenic rate of EJ cells transfected with rAAV-ES was only 2/5 of that of the control group. The in vivo experiments confirmed that long-term and high-efficiency of endostatin in serum after systemic administration of rAAV-ES (30-40 ng / ml), slow tumor growth (32 d ± 9 d) and low microvessel density [(8 ± 3) /0.739 mm2). The difference was statistically significant Significance (all P <0.05), no ischemic and other abnormal changes in brain and brain histological examination. Conclusions rAAV-ES has no toxic and side effects, which can effectively inhibit tumor angiogenesis and thus inhibit the occurrence and development of bladder cancer. The successful packaging of rAAV-ES laid the foundation for the in situ gene therapy of bladder cancer.