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

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

利用基因重组技术，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重组后转杂至病毒包装细胞，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，二周后计数存活的最性细胞克隆数，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。

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.