摘要
目的 :构建hTM真核表达质粒 ,并导入脐静脉内皮细胞 (HUVECs)中表达 ,为临床血栓病的基因治疗提供实验依据和物质基础。方法 :PCR法扩增hTM基因的全长表达片段 ,HindⅢ /EcoRⅠ酶切后与pcDNA3 1(+) /neo质粒连接成pcDNA3 1/hTM。经鉴定正确后 ,由阳离子脂质体包裹转染HUVECs ,免疫组化检测转染后细胞hTM的表达。结果 :双酶切及测序鉴定均证实hTM基因被成功克隆。pcDNA3 1/hTM经脂质体介导能有效地转染内皮细胞 ,转染效率约为 10 %左右。结论 :成功构建pcDNA3 1/hTM重组质粒 ,并且能在内皮细胞中高效表达。
AIM: To provide experimental evidence for gene therapy of thrombophilia disease, we constructed the eukaryotic expression plasmid with human thrombomodulin (hTM) gene and observed the alteration of hTM expression on the surface of human umbilical vein endothelial cells (HUVECs) with and without the reconstructive plasmid. METHODS: The whole expressive fragment of hTM gene was amplified by PCR from human genome. Both hTM gene and pcDNA3.1(+)/neo empty vector was digested by HindⅢ and EcoRⅠ. Two digested fragments were ligated into pcDNA3.1/hTM with T_4DNA ligase. After identifying, the reconstructive plasmid transfected into HUVECs using lipofectin. The hTM antigen on the HUVECs was detected by immunohistochemistry. RESULTS: The hTM reconstructive plasmid was confirmed by double endonuclease redigesting and sequencing. About 10% HUVECs were transfected by pcDNA3.1/hTM plasmid with lipofectin and the high-level hTM was detected on the transfected cells. CONCLUSION: We constructed the pcDNA3.1/hTM plasmid successfully, and it could be expressed on the HUVECs. [
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2004年第7期1200-1203,共4页
Chinese Journal of Pathophysiology
关键词
血栓调节蛋白
脐静脉
内皮细胞
质粒
Thrombomodulin
Umbilical veins
Endothelial cells
Plasmids
