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目的探索一条肿瘤特异性基因治疗的新途径。方法将新型人 TNF- α D11a基因和 KDR特异性启动子插入到 3’L TR缺失 2 99bp的逆转录病毒载体 p L XSN中 ,构建成 p L XSN- D2 99- KDRp- TNF- α D11a重组逆转录病毒载体 ,使目的基因转录受 KDR启动子驱动。通过脂质体介导将该重组载体转染 PA317包装细胞 ,并用病毒上清感染血管内皮细胞和NIH3T3细胞。分别经 EL ISA和 MTT法测定 TNF- α D11a在血管内皮细胞中的表达及其生物学活性。结果成功构建了携带 TNF- α D11a和 KDR启动子的逆转录病毒载体 ,TNF- α D11a在血管内皮细胞中的表达水平及细胞增殖抑制作用均高于NIH3T3细胞。结论 KDR启动子可使外源 TNF- αD11a在血管内皮细胞中特异性表达
Objective To explore a new approach to tumor-specific gene therapy. METHODS: The novel human TNF-α D11a gene and KDR-specific promoter were inserted into the retroviral vector p L XSN with a 3′ L TR deletion of 299 bp to construct a p L XSN-D2 99-KDRp-TNF-α D11a recombination. The retroviral vector allows transcription of the gene of interest to be driven by the KDR promoter. The recombinant vector was transfected into PA317 packaging cells via liposome-mediated transfection, and the virus supernatant was used to infect vascular endothelial cells and NIH3T3 cells. The expression and biological activity of TNF-α D11a in vascular endothelial cells were determined by EL ISA and MTT methods, respectively. Results The retroviral vector carrying TNF-α D11a and KDR promoter was successfully constructed. The expression level of TNF-α D11a in vascular endothelial cells and the inhibitory effect of cell proliferation were higher than NIH3T3 cells. Conclusion The KDR promoter can specifically express exogenous TNF-αD11a in vascular endothelial cells.