应用端粒酶逆转录酶基因使猪血管内皮细胞永生化的初步研究

Elementary Study on Immortalization of Porcine Vascular Endothelial Cell by Telomerase Reverse Transcriptase Transfection

为寻求使猪主动脉血管内皮细胞增殖寿命延长的方法,本试验将含有人类端粒酶催化亚基端粒酶逆转录酶(Human telomerase reverse transcriptase,hTERT)基因的真核表达质粒pCI-neo-hTERT通过脂质体介导法导入猪主动脉血管内皮细胞(Porcine arterial endothelial cell,PAEC)。对转染后的血管内皮细胞进行Ⅷ因子和CD34鉴定,利用PCR-ELISA方法检测hTERT导入后细胞端粒酶活性的表达情况,探讨了转染后细胞的生长特性。结果显示,转染后的细胞Ⅷ因子和CD34均呈阳性,证明转染后细胞仍表达血管内皮细胞的特异性蛋白;导入hTERT后,细胞有较高的端粒酶活性,增殖寿命较对照细胞延长了20代。表明hTERT导入细胞能重建细胞端粒酶活性,从而延长细胞在体外培养的寿命。

To elongate the proliferation life-span of porcine arterial endothehal cell（PAEC） in vitro. The eukaryotic expression plasmid pCI-neo-hTERT was transfected into PAEC by lipofection. FactorⅧ related antigen and CD34 of PAEC were identified after transfection by immunohistochemical method; we also detected telomerase activity of PAEC after transfection by PCR- ELISA, The results showed that Factor Ⅷ related antigen and CD34 in PAEC was positive after transfection. The PAEC after transfection expressed the vascular endothelial cell-specific protein still; Telomerase activity in PAEC was higher by hTERT introduction, the cell proliferation lifespan was elongated to 20 population doublings compared to control. Our research indicated that telomerase activity can be activated in PAEC, thus the cell proliferation life-span was elongated in vitro.