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目的 :为进行TNFR的靶向基因治疗研究打基础。方法 :将人TNFR55和TNFR75基因序列分别与KDR启动子 (KDRp)及灭活自身启动子的逆转录病毒载体RV D2 99重组 ,构建RV D2 99 KDRp TNFR55及RV D2 99 KDRp TNFR75逆转录病毒载体 ,分别转染包装细胞PA31 7,获得稳定的产病毒细胞系。 结果 :获得的TN FR55和TNFR75产毒细胞系病毒滴度分别为 1× 1 0 5CFU ml和 2× 1 0 5CFU ml。感染RV D2 99 KDRP TNFR55病毒的ECV30 4细胞与TNF孵育后的培养上清 ,对L92 9细胞的细胞毒活性比相同条件下的NIH3T3细胞低约2 6倍 ,而感染RV TNFR55病毒的ECV 30 4与相同条件下的NIH3T3细胞无明显差异。结论 :建立了TNFR55和TNFR75逆转录病毒高产毒细胞系 ,构建的RV D2 99 KDRP TNFR55和RV D2 99 KDRP TNFR75重组病毒载体可以介导TNFR在血管内皮细胞中靶向表达
OBJECTIVE: To lay the foundation for the study of targeted gene therapy for TNFR. METHODS: The human TNFR55 and TNFR75 gene sequences were recombined with the KDR promoter (KDRp) and the retroviral vector RV D299, respectively, to inactivate the self promoter. RV D2 99 KDRp TNFR55 and RV D2 99 KDRp TNFR75 retroviral vectors were constructed, Transfection of packaging cells PA31 7, to obtain a stable virus-producing cell lines. Results: The titers of TN FR55 and TNFR75 producing virus cell lines were 1 × 10 5 CFU ml and 2 × 10 5 CFU ml, respectively. ECV304 cells infected with RV D299 KDRP TNFR55 virus The culture supernatant incubated with TNF had a cytotoxic activity on L92 9 cells about 26-fold lower than that of NIH3T3 cells under the same conditions, while ECV 30 4 infected with RV TNFR55 virus No significant difference with NIH3T3 cells under the same conditions. Conclusion: The TNFR55 and TNFR75 retrovirus high producing cell lines were established, and the recombinant viral vectors RV D2 99 KDRP TNFR55 and RV D2 99 KDRP TNFR75 could induce the target expression of TNFR in vascular endothelial cells