重组腺相关病毒介导内皮抑素的表达及体外活性研究

Adeno-associated virus-mediated expression of human endostatin and its biological activity in vitro

目的构建携带人内皮抑素基因的重组腺相关病毒rAAV-hEndo,介导其体外表达并检测其生物活性。方法采用无需包装辅助病毒的双质粒共转染方法制备rAAV-hEndo,测定其滴度及感染效率。免疫荧光染色检测内皮抑素蛋白在体外感染细胞中的表达,并通过MTT法、细胞周期检测及TUNEL法检测细胞凋亡指数,观察其对人脐静脉内皮细胞ECV304的影响。结果所制备的重组腺相关病毒滴度为2×1012vg/ml,感染效率达98%。免疫荧光染色显示内皮抑素蛋白主要表达于细胞胞质。rAAV-hEndo感染细胞培养上清对ECV304细胞72h增殖抑制率为67.3%。内皮细胞转染内皮抑素基因后细胞周期明显阻滞于G1期,其G1期占(72.5±4.0)%,与对照组(52.1±2.1)%比较有显著差异(P<0.01)。TUNEL法检测实验组内皮细胞凋亡指数为(32.6±3.2)%,与对照组(4.2±1.9)%比较有显著差异(P<0.01)。结论所制备的重组腺相关病毒rAAV-hEndo能有效介导具有生物活性的内皮抑素表达,为进一步肿瘤抗血管生成基因治疗动物实验奠定了基础。

Objective To construct the recombinant adeno-associated viral vector containing human endostatin gene (rAAV-hEndo) and observe the biological activity of the expressed human endostatin in vitro. Methods rAAV-hEndo was prepared using a helper virus-free packaging system. The rAAV viral genome titer was quantified by Taqman real-time PCR, and the endostatin expressed in human umbilical vein endothelial cell line ECV304 was detected by immunofluorescence staining. The effects of endostatin on ECV340 cells were evaluated by MTT cell proliferation assay, cell cycle analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) technique. Results The viral titer of rAAV-hEndo prepared was 2×1012 vg/ml and the vector had an infection efficiency of 98%. Immunofluorescence staining showed that the humam endostatin protein was expressed mainly in the cytoplasm of ECV304 cells, and the proliferation of the cells was obviously inhibited by the supernatant of rAAV-hEndo, with a inhibition rate of 67.3% 72 h after the addition of the supernatant. ECV304 cells infected with rAAV-hEndo were obviously arrested in G1 phase, and the G1-phase cell percentage of treatment group were significantly higher than that of control group [(72.5±4.0)% vs (52.1±2.1)%, P<0.01]. ECV304 cells infected with rAAV-hEndo demonstrated markedly enhanced apoptosis, with a significantly greater apoptotic index than that of the control cells [(32.6±3.2)% vs (4.2±1.9)%, P<0.01]. Conclusion rAAV-hEndo can effectively mediate the expression of biologically active human endostatin, which may facilitate further study of antiangiogenic gene therapy with endostatin for cancers.