人肿瘤抑素慢病毒表达载体在肝癌细胞中的表达

Expression of human tumstatin in heptocarcinoma cell and identification of its biological funtions

目的:观察人肿瘤抑素(tumstatin)对血管生成的抑制作用,探讨肿瘤抑素在肝癌基因治疗中的应用前景。方法:构建人肿瘤抑素慢病毒表达载体pLenti-Tum,经293T细胞包装,收集病毒上清液,感染肝癌细胞株SMCC-7721,收集各组细胞的条件培养基,通过蛋白质印迹分析各组细胞肿瘤抑素的表达情况,并通过人脐静脉血管内皮细胞增殖抑制及迁移试验研究其体外生物学活性。结果:限制性内切酶酶切分析和测序结果均表明成功构建了肿瘤抑素慢病毒表达载体,以293T细胞包装的重组慢病毒能够高效感染肝癌细胞,肝癌细胞感染重组慢病毒后可高效表达肿瘤抑素并分泌到培养上清液中。体外研究结果表明,重组肿瘤抑素可明显抑制人脐静脉血管内皮细胞增殖及迁移,抑制率分别达35%和41%,与空白对照组相比,差异有统计学意义(P值均<0.01)。结论:肿瘤抑素慢病毒表达载体在SMCC-7721细胞中能获得稳定表达,并且重组肿瘤抑素能有效遏制肿瘤的血管生成。

Objective： To evaluate the antiangiogenic property of vascular endothelial cell in heptocarcinoma cell lines and explore its possible application in the gene therapy of human hepatocellular carcinoma（HCC）.Methods： The gene encoding tumstatin was subcloned into lentiviral vector to generate the recombinant vector pLenti-Tum.The vector pLenti-Tum and other packaging vector were contransfected to 293T cells by calcium phosphate.Then SMCC-7721 was infected with recombinant lentivirus and the expression efficiency of tumstatin was analyzed by western blot.Proliferation and migration assay of human umbilical vein endothelial cells（HUVEC） was used to evaluate the biological activity of tumstatin in vitro.Results： Restriction enzyme digestion and DNA sequencing demonstrated that the recombinant plasmid Plenti-Tum was constracted.SMCC-7721 secreted tumstatin in the media effectively after infected with the recombinant lentivirus and this protein exhibited strong inhibitory effects on proliferation and mirgration of HUVEC（P0.01）.Conclusion： SMCC-7721 secreted tumstatin effectively after infected with recombinant lentivirus and this recombinant tumstatin may exert inhibitory on tumor antiangiogenic.