肝型丙酮酸激酶启动子与人胰岛素基因逆转录病毒介导感染pT67、HepG2细胞的蛋白质表达

Protein expression of pT67 and HepG2 cells spin-infected by retrovirus with LPK promoter and human insulin gene

目的检测新构建肝型丙酮酸激酶启动子(LPKp)与人胰岛素基因(hInsg)逆转录病毒表达载体(pM54,LPKp-hInsg)在pT67、HepG2细胞的表达情况,为基因治疗或基因结合干细胞治疗糖尿病寻找新的优化生物载体。方法 (1)限制酶切父、母本质粒p54、pMDN-SIN,取相关片段构建含人Ins基因-逆转录病毒载体pM54(pMDN-SINq-p54;LPKp-hInsg);pM54扩增、纯化、酶切鉴定;(2)脂质体FuGENE 6转染pM54质粒进入pT67、Phoenix E和3T3细胞系,同时转染pCMVβGal和父本质粒p54做对照;(3)制备转染后细胞培养拟逆转录病毒上清液;(4)用转染后细胞培养上清液旋转感染pT67、HepG2细胞;(5)ELISA法检测转染、感染后细胞培养上清液Ins含量。结果酶切鉴定质粒与构建预期相符;ELISA法检测出转染、感染上清液中均含较高水平Ins。对照结果均为阴性。结论 LPKp-hInsg基因逆转录病毒表达载体pM54构建成功。人胰岛素基因逆转录病毒介导旋转感染pT67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奠定了基础。

Objective To check the protein expression of pT67 and HepG2 cells spin-infected by retrovirus with human insulin gene （hlNSg） and LPK promoter （livertype pyruvate kinase promoter, LPKp） for gene or stem cell therapy of diabetes. Methods （1）Cutting the vector and inserting the parent （pMDN-SIN, p54） plasmids with restriction enzyme were to make pM54 （LPKp-hINSg）. The plasmid pM54 was expanded and identified. （2）The pM54, p54 and pCMVβGal plasmids were transfected into Phoenix E and pT67 package cell lines by FuGENE Method. The 3T3 cell was used as control. （3） Supernatants were made and collected after transfected for 48 or 72 hours with DNA. （4） The pT67 and HepG2 cells were spin-infected by retrovirus supernatants collected at 72 hours infection. （5）The expression of INS was checked by ELISA. Results The retroviral vector pM54 was successfully constructed. The human insulin gene expression was upregulated in supernatants after the pT67 and HepG2 cells were infected. Conclusions The expression of the human insulin gene in HepG2 and pT67 cells provides a vital data for the further research of the insulin gene elements modified to intervene the stem cell insulin production to treat diabetes.