基础医学与临床 ›› 2007, Vol. 27 ›› Issue (11): 1241-1245.

• 研究论文 • 上一篇    下一篇

兔外周血内皮祖细胞培养及tHK 基因体外修饰的研究

刘春喜 李大庆 张运 姜虹 王荣 王旭平   

  1. 山东大学齐鲁医院心内科/心血管重构与功能研究教育部重点实验室 山东大学齐鲁医院心内科 北京大学第一医院肾内科
  • 收稿日期:2006-09-24 修回日期:2007-01-16 出版日期:2007-11-25 发布日期:2007-11-25
  • 通讯作者: 李大庆

Study of culture and tHK gene modification of endothelial progenitor cells from rabbit peripheral blood

ChunXi Liu LI Da-Qing Yun Zhang Hong Jiang Rong Wang XuPing Wang   

  • Received:2006-09-24 Revised:2007-01-16 Online:2007-11-25 Published:2007-11-25
  • Contact: LI Da-Qing

摘要: 内皮祖细胞(endothelial progenitor cells ,EPCs )的繁殖能力和归巢特性提示其作为基因转移载体的良好潜力,但能否在体外被修饰并有效表达目的基因,是基因修饰EPCs双重治疗血管病的技术策略能否实现的前提条件。本文从兔外周血中分离鉴定内皮祖细胞(endothelial progenitor cells ,EPCs ),PCR扩增人激肽释放酶(Kallikrein,tHK)目的基因,将该全长基因定向克隆至真核表达载体pEGFP-N3上,构建以绿色荧光蛋白(Green fluorescence protein ,GFP) 为报告基因的重组真核表达载体,并利用脂质体转染体外培养的EPCs,在活细胞状态下用荧光显微镜直接观察tHK-EGFP融合蛋白在细胞中的表达;用RT-PCR 方法验证tHK在mRNA水平的表达;用ELISA方法验证tHK蛋白水平的表达。结果表明,通过基因克隆方法成功构建了pEGFP-Kallikrein重组质粒载体,并且在EPCs 中稳定表达,表达的融合蛋白具有tHK 和EGFP 的双重活性,tHK基因修饰EPCs对于血管病治疗具有良好的前景。

关键词: 激肽释放酶, 内皮祖细胞, 动脉损伤, 血管病

Abstract: Abstract:To construct pEGFP-Kallikrein eukaryotic expression vector which used green fluorescence protein as reportor gene, and transfect rabbit endothelial progenitor cells. The human tissue kallikrein(tHK)gene were amplified with PCR, and inserted into pEGFP-N3 vector. The rabbit endothelial progenitor cells(EPCs) were transfected with the formed plasmid by means of lipidosome, and the kallikrein-EGFP fused protein was visued directly with fluorensce microscope, and the expression of tHK was detected by RT-PCR(reverse transcription-PCR) and ELISA. Plasmids was formed correctly and detected by PCR meanwhile being sequenced, and expressed in the rabbit EPCs. The fused protein has actitivites of both kallikrein and EGFP. The tHK genetically modified EPCs could play a role in the treatment of artery injury and endothelial dysfunction.

Key words: Kallikrein, Endothelial progenitor cells, Artery injury, Vascular disease