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应用基因重组技术构建了人可溶性FL基因逆转录病毒载体pLXSN FL ,经转染PA317细胞和G4 18筛选抗性细胞克隆 ,获得重组病毒液。NIH3T3细胞进行病毒滴度测定后 ,选择适当滴度的重组病毒 (5× 10 5CFU/ml)感染ECV30 4细胞 ;应用聚合酶链反应 (PCR )及反转录聚合酶链反应 (RT PCR )检测感染ECV30 4细胞FL的DNA及mRNA表达 ;应用ELISA测定rhFL在ECV30 4中的表达水平 ,用荧光标记和流式细胞仪检测转基因上清对单个核细胞来源DC的表型影响及3 H TdR掺入法测定DC对T细胞的促增殖作用。结果表明 ,外源性rhFL基因整合到ECV30 4细胞染色体DNA并有效地转录和翻译 ,稳定表达水平为 82 4ng (10 6细胞 /2 4h )的人FL。转基因ECV细胞培养上清能有效诱导人PBMC来源DC的分化和增强DC对T细胞的激发作用。外源性FL基因可以转移到ECV30 4细胞并稳定表达 ,有助于体外研究内皮细胞与免疫细胞的相互作用
The retroviral vector pLXSN FL of human soluble FL gene was constructed by gene recombination technique. The transfected PA317 cells and G4 18 resistant cell clones were selected to obtain recombinant virus liquid. NIH3T3 cells were infected with recombinant virus (5 × 10 5 CFU / ml) and then transfected into ECV30 4 cells with appropriate titer of virus titer. Polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT PCR) The expression of DNA and mRNA of FL in ECV304 cells was detected by ELISA. The expression of rhFL in ECV30 4 was detected by ELISA. The phenotypic effects of transgenic supernatants on DCs derived from mononuclear cells were detected by fluorescence labeling and flow cytometry. Into the law to determine DC on T cell proliferation. The results showed that the exogenous rhFL gene was integrated into the chromosomal DNA of ECV30 4 cells and efficiently transcribed and translated into human FL with a stable expression level of 82 4 ng (10 6 cells / 24 h). The supernatant of transgenic ECV cells can effectively induce the differentiation of human PBMC-derived DCs and enhance the stimulating effect of DC on T cells. Exogenous FL gene can be transferred to ECV304 cells and stably expressed, helping to investigate the interaction of endothelial cells and immune cells in vitro