兔外周血内皮祖细胞培养及HTK基因体外修饰  

Culture of endothelial progenitor cells from rabbit peripheral blood and HTK gene modification

作　　者：刘春喜[1] 李大庆[1] 张运[1] 姜虹[1] 王荣[1] 王旭平[1] 

机构地区：[1]山东大学齐鲁医院心内科教育部和卫生部心血管重构和功能研究重点实验室,山东济南250012

出　　处：《基础医学与临床》2007年第11期1241-1245,共5页Basic and Clinical Medicine

基　　金：山东省科技厅优秀中青年科学家基金(9915)

摘　　要：目的探讨兔外周血来源的内皮祖细胞(EPCs)经体外基因修饰后能否成功表达目的基因。方法分离兔外周血单个核细胞进行定向培养,观察细胞摄取DiI-Ac-LDL的能力,免疫荧光法检测EPCs的细胞表面标记。同时PCR扩增人组织激肽释放酶(HTK)基因,构建以GFP为报告基因的重组真核表达载体,转染体外培养的EPCs后,荧光显微镜下观察HTK-EGFP融合蛋白的表达,通过RT-PCR与ELISA方法检测HTK的表达水平。结果细胞培养5～6d开始出现成簇的短梭形贴壁细胞,15d后形成鹅卵石样细胞层,传代后的细胞能摄取DiI-Ac-LDL,免疫荧光显示vWF和CD133+;pEGFP-HTK重组质粒在EPCs中可以稳定表达,表达的融合蛋白具有HTK和EGFP的双重活性。结论HTK基因修饰兔外周血EPCs对于血管病的治疗可能具有良好的前景。Objective To investigate the feasibility of gene modification of peripheral blood derived endothelial progenitor cells（EPCs）. Methods The rabbit mononuclear cells were collected from rabbit peripheral blood and cultured. The uptaking test of DiI-Ac-LDL was directly visualized with fluorescent microscopy, the immunofluorescence analysis was performed to detect the expression of surface marker. And the human tissue kallikrein（HTK） gene was amplified with PCR, and inserted into pEGFP-N3 vector. EPCs were transfected with the constructed plasmid by means of lipidosome, and the HTK-EGFP fused protein was visualized directly with fluorescent microscopy, and the expression of HTK was detected by RT-PCR and ELISA. Results After 5 - 6 days of culture, spindle-shaped attached cells clustered together, and cobblestone-like cell layer appeared after 15 days. Cells were positive of uptaking DiI-Ac-LDL and expressing vWF and CD133 related antigen. Plasmids were correctly formed and expressed in the rabbit EPCs. The fused protein had activity of both HTK and EGFP. Conclusion The HTK gene modified EPCs is potential in the treatment of artery injury and endothelial dysfunction.

关 键 词：激肽释放酶 内皮祖细胞 动脉损伤 血管病 

分 类 号：Q954.563[生物学—动物学]