基因-病毒治疗系统CNHK200-hA的构建及体外初步研究

目的　构建一种结合抗肿瘤新生血管生成的基因治疗与病毒治疗优势的新型肿瘤基因 病毒治疗系统CNHK2 0 0 hA。方法　克隆人抗血管生成基因Angiostatin(k1 5 ) ,命名为hA。利用病毒重组技术将hA插入肿瘤特异性增殖病毒的病毒基因组中 ,通过病毒增殖试验、电镜技术、Westernblot分析、鸡胚尿囊膜试验 (CAM ) ,观察CNHK2 0 0 hA的复制情况、hA基因的表达及其对新生血管生成的抑制作用。结果　构建了一种新型的基因 病毒治疗系统CNHK2 0 0 hA。该治疗系统为E1b 5 5× 10 3 蛋白缺失而保留了腺病毒E1a蛋白的 5型腺病毒 ,能在肿瘤细胞内大量增殖及复制 ,而在正常细胞内增殖能力较弱 ,从而特异性杀灭肿瘤细胞。同时CNHK 2 0 0 hA携带人抗肿瘤新生血管生成基因hA ,能在肿瘤细胞内高效表达hA蛋白 ,其表达量高于传统基因治疗的腺病毒载体系统。电镜证实该治疗系统可在肺癌A5 49细胞株中复制及增殖。CAM试验证实该治疗系统感染肺癌细胞后的培养液上清具有抗新生血管生成的生物活性。结论　基因 病毒治疗系统CNHK2 0 0 hA能在肿瘤细胞中增殖复制 ,并明显提高hA基因的表达量 ,表达产物具有抑制新生血管形成的作用。

Development of gene-viral therapeutic system CNHK200-hA and its primary study in vitro

Objective To develop a novel gene-viral therapeutic system which combines the advantages of the gene therapy,antiangiogenic therapy and virotherapy.Methods The antiangiogenic gene,designated human angiostatin (k1-5,hA),was inserted into the genome of the replicative virus specific for the tumor cells by virus recombination technology.The replication of the virus,the expression of the antiangiogenic gene of hA and the suppression of angiogenesis were observed respectively by virus replication assay in vitro,electron microscopy,Western blot,and chicken embryo chorioallantoic membrane (CAM) assay.Results A new kind of gene-viral vector system,designated CNHK200-hA,in which the E1b55000 gene was deleted but the E1a gene of adenovirus was preserved,was constructed.The novel vector system possessed the same property as the replicative virus ONYX-015,replication and proliferation in the p53 - tumor cells and in some p53 tumor cells but not in the normal cells,thus specifically killing the tumor cells.Besides,it carried the antiangiogenic gene,human angiostatin (k1-5),further enhancing the effect of anti-tumor.When carrying the gene of hA,it made the expression of this gene in tumor cells far more effectively than the adenovirus vector employed in the traditional gene therapy.Electron microscopy confirmed that this vector system replicated and proliferated in the lung tumor cells.CAM assay demonstrated that the hA produced by CNHK200-hA infected tumor cells possessed antiangiogenic bioactivity.Conclusion CNHK200-hA,a novel vector in which the antiangiogenic gene is inserted into the genome of the replicative virus specific for the tumor cells,can increase the expression level of antiangiogenic gene.It possesses all the advantages of gene therapy,virotherapy,and antiangiogenic therapy,thus further enhancing the curative effect and overcoming such disadvantages as low transfer rate,low expression,lack of target tropism and low anti-tumor activity.