人组织型纤维蛋白溶解酶原激活剂cDNA在牛血管内皮细胞中的表达

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．

Expression of Human Tissue-type Plasminogen Activator cDNA in Endothelial Cell of Bovine

Aim To determine the expression of human tissue-type plasminogen activator (tPA) cDNA in mamalian cells. Methods A recombinant retroviral vector containing tPA gene was constructed and transtected into bovine endothelial cells (EC) by retroviral vector-mediated gene transfer. The tPA activity of transduced cells was measared with casein-plastninogen-agarose plate and a synthetic-peptide substract S-2390.Results The viral titer was 4 ×108 cfu/L. Trans-fection effciency was 6×108 colonies/g DNA/106 cells. The tPA activity of transduced cells was obvi-ously increased. Immunohistochemistry analyses confirmed the expression of tPA gene in the genetically engineered cells.Conclusions Retroviral vector mediated gene trans-fer can be used to enhance the fibrinolytic activity of EC. It might be an approach for tPA gene therapy.