论文部分内容阅读
利用基因生组技术，将人组织型纤维蛋白溶解酶原激活剂cDNA与逆转录病毒载体LNSX重组后转染至病毒包装细胞．其分泌的重组逆转录病毒颗粒再用来感染NIH3T3细胞和牛血管内皮细胞，经G418筛选，二周后计数存活的阳性细胞克隆数，得到病毒滴度为4×10~8cfu／L。计算转染效率为6×10~8克隆/gDNA/10~6细胞。在含人纤维蛋白原和凝血酶的纤维蛋白板上点样并用标准品作对照，发现经逆转录病毒感染的重组内皮细胞和包装细胞．分泌的细胞培养液，在纤维蛋白板上显示有大小不一的溶圈。使用发包底物法也测得含人组织型纤维蛋白溶解酶原激活刘cDNA转基因内皮细胞培养液的分泌活性．正常的内皮细胞则由于分泌量少而在敏感度以下．使用特异性的免疫组织化学杂色并经图像分析证实，人组织型纤维蛋白溶解酶原激活剂基因在牛的血管内皮细胞中得到表达。说明人组织型纤维蛋白溶解酶原激活剂基因的cD-NA已正确转入牛的血管内皮细胞中，为将来进行基因治疗展示了良好的应用前景．
Recombinant human tissue-type plasminogen activator cDNA was recombined with the retroviral vector LNSX and transfected into virus-packaged cells using gene engineering techniques. The secreted recombinant retroviral particles were used to infect NIH3T3 cells and bovine vascular endothelial cells, and screened by G418. After two weeks, the number of surviving positive cell clones was counted, and the virus titer was 4 × 10-8 cfu / L. The transfection efficiency was calculated as 6 × 10 ~ 8 clones / gDNA / 10 ~ 6 cells. Samples were spotted on fibrin plates containing human fibrinogen and thrombin and used as controls to identify retroviral infected recombinant endothelial cells and packaging cells. Secreted cell culture medium, in the fibrin plate shows the size of the dissolution circle. Secretion activity of culture media containing human tissue-type plasminogen activator rhizobial cDNA transgene endothelial cells was also measured using the hairpin substrate assay. Normal endothelial cells are less sensitive due to less secretion. Using specific immunohistochemical stains and confirmed by image analysis, the human tissue-type plasminogen activator gene was expressed in bovine vascular endothelial cells. This indicated that cD-NA of human tissue-type plasminogen activator gene has been correctly transferred into bovine vascular endothelial cells, which has shown a promising future for gene therapy.