TNFa在血管内皮细胞ECV304中的靶向表达研究

在缺失了3'LTR U3区内病毒的启动子／增强子序列的逆转录病毒载体pLXSNd中,用血管内皮生长因子受体KDR的特异性启动子调控了TNFa在血管内细胞ECV304中的靶向表达。将构建的载体pLXSN-TNFa,pLXSNd-KDRp-TNFa和空载体pLXSN用PA317细胞包装后获得重组病毒,并用重组病毒分别感染NIH3T3细胞和ECV304细胞,培养物上清的ELISA结果证明,KDR启动子指导的TNFa在KDR阳性细胞ECV304中的表达量为在KDR阴性细胞NIH3T3中的表达量的8倍;而TR指导的TNFa在这两种细胞中的表达无明显差异,实现了TNFa在血管内皮细胞中的靶向表达,这可能为肿瘤基因治疗提供新途径。

Targeted expression of TNF a in ECV304 cells

Results presented in this paper show that TNF a had been specifically expressed in vascular endothelial cells under the control of KDR p in pLXSN whose promoter and enhancer were deleted. Plasmids pLXSN-TNF a and pLXSN d-KDR p-TNF a were constructed and transfected into package cell line PA317 using Lipofectin AMINE method. The supernatant containing the virus was detected with NIH3T3 and was used to transfect NIH3T3 and ECV304 cells. After selection by G418, G418 resistant cell clones were cultured. ELISA analysis of the supernatant collected from tranfected clones showed that TNF-a expression level in ECV304 under the control of KDR p was 8 fold higher than that in NIH3T3, but TNF a expression level under the retroviral promoter had no difference between NIH3T3 and ECV304. The results strongly suggested that TNF a gene in the plasmid pLXSN d-KDR p-TNF a was specifically expressed in ECV304 cells under the control of KDR p. Therefore, this study illustrated the superiority of using targeted expression vectors in cancer gene therapy .