人胰岛素基因逆转录病毒旋转感染Phoenix细胞的表达

Expression of phoenix cells were spin-infected by retrovirus with human insulin gene

目的:观察新构建人胰岛素基因逆转录病毒表达载体(pM54)在Phoenix和HepG2等细胞的表达情况。方法:1)脂质体法转染pM54质粒进入pT67、Phoenix和3T3细胞系;2)用新构建质粒的父本质粒p54和pCMVβGal质粒做转染基因对照;3)制备转染后细胞培养拟逆转录病毒上清液;4)旋转感染Phoenix及HepG2细胞;5)ELISA法检测转染、感染后上清液INS含量。结果:ELISA法检测证实感染细胞培养上清液中含较高水平目的蛋白INS的表达。对照结果均为阴性。结论:人胰岛素基因逆转录病毒介导旋转感染Phoenix等细胞,目的蛋白INS获得了预期的表达。实验为基因治疗或基因结合干细胞治疗糖尿病的进一步相关研究提供了重要数据。

Objective： To investigate the expression of Phoenix and HepG2 cells which were spin-infected by retrovirus with human insulin gene. Methods： To transfect the pM54, p54 and pCMVβGal plasmids into pT67 and Phoenix package cell lines by FuGENE Method; 3T3 cell was control; Supematants were collected after transfection 48 or 72 hours with DNA; Spin-infected Phoenix and HepG2 cells by retrovirus; supematants were collected at 72 hours post-infection; Check the expression of INS by ELISA. Results： ELISA method detecting proved that the human insulin protein was expressed in the supernatants of spininfected HepG2 and Phoenix cells. All the controls were negative. Conclusion： This study provide a vital data for the further research of the insulin gene elements, which was modified to intervene the stem cell insulin production to treat diabetes related subject.