人脐动脉平滑肌细胞培养及转染TFPI基因的实验研究

目的 探讨组织因子途径抑制因子（TFPI）基因对人脐动脉血管平滑肌细胞生长的影响,为TFPI基因用于血管再狭窄的治疗提供理论依据和实验基础.方法 从人脐动脉分离平滑肌细胞,通过免疫组化方法进行细胞鉴定;用不同剂量pIRES-TFPI基因（分别为1,2,3 μg/mL）转染血管平滑肌细胞,采用RT-PCR测定细胞内TFPI表达以优化基因转染条件;通过MTT法测定TFPI基因对人脐动脉血管平滑肌细胞生长的影响.结果 分离得到的血管平滑肌细胞的纯度高于90%;3个剂量的基因转染后,细胞内TFPI基因的表达水平无明显差异.采用2 μg/mL转染剂量时,TFPI基因转染后第5天,脐动脉血管平滑肌的生长受到明显抑制.结论 通过基因转染的方式将TFPI基因导入细胞对人脐动脉平滑肌的增殖具有抑制作用.

Isolation and culture of human umbilical artery smooth muscle cells and gene transfection of TFPI gene

Objective The aim of the present study is to investigate the effect of tissue factor pathway inhibitor (TFPI) gene on cell cycle of human vascular smooth muscle cells.Methods Human vascular smooth muscle cells were separated from human umbilical artery and identified by immunohistochemical staining.The cells were transfected with various amount of pIRES-TFPI plasmid (1,2,and 3 μg/ml,respectively)and the TFPI expression in the cells were analyzed by RT-PCR.MTT assay was employed to detect the effect of TFPI gene on the proliferation of human vascular smooth muscle cells.Results The proliferation of vascular smooth muscle cells was inhibited in pIRES-TFPI group 5 and 7 days after gene transfection when compared with that of pIRES 1-neo transfection group.Conclusion The overexpression of TFPI gene in human umbilical artery vascular smooth muscle cells may contribute to the suppression of the proliferation of cells by gene transfection.