大鼠胰岛素原基因转染肠道上皮细胞构建类胰岛B细胞系的研究

Construction of Pseudo-B Cell Line by Transfecting Rat Proinsulin Gene to Mouse Intestinal Epithelial Cell

目的观察重组大鼠胰岛素原基因转染小鼠肠道上皮细胞(IEC-6)后,IEC-6的胰岛素表达情况。方法将IEC-6分为重组大鼠胰岛素原基因pCMV/proinsulin转染组(A组)、空载质粒pCMV转染组(B组)和未转染组(C组)。利用脂质体法将pCMV/proinsulin和pCMV分别转染IEC-6,用RT-PCR法检测转染后IEC-6胰岛素原基因mRNA的表达,并用超敏放免法测定转染后IEC-6的胰岛素表达水平。结果pCMV/proinsulin转染IEC-6后,IEC-6能表达胰岛素原基因mRNA。3组IEC-6表达的胰岛素水平间差别有统计学意义(P<0.05)。A组IEC-6胰岛素表达水平与B组、C组比较,差别有统计学意义(P<0.001);B组IEC-6胰岛素表达水平与C组比较,差别无统计学意义(P>0.05)。结论重组大鼠胰岛素原基因能成功转染小鼠肠道细胞并分泌胰岛素,为开展糖尿病基因治疗的研究奠定了实验基础。

Objective To investigate the expression of rat intestinal epithelial cells （ IEC - 6） insulin after construction of pseudo - line by transfecting rat proinsulin gene to mouse IEC - 6. Methods IEC - 6 were divided into three groups ： Group A with recombinant rat proinsulin gene （pCMV/proinsulin） , Group B with no -load plasmid （pCMV） and Group C without transfection. The pCMV/proinsuin and pCMV were transfected to IEC - 6 by lipofetin - mediated DNA transfection. RT - PCR was used to determine expression of proinsuin gene mRNA in modified IEC - 6 of insulin gene, and hypersensitive radioimmunity was applied to test insulin expression in IEC -6 after transfection. Results After transfection , proinsulin gene mRNA could be detected in IEC - 6. There was significant difference in insulin level among the three groups （ P 〈 0. 05 ） ; Group A was significantly different from Group B and Group C in expression of IEC - 6 insulin （ P 〈 0. 001 ） ; No significant difference was observed in expression of IEC - 6 insulin between Group B and Group C （ P 〉 0. 05 ）. Conclusion Recombinant rat proinsulin gene can be successfully transfected to mouse IEC - 6 and excrete insulin. It is a laboratory base for study of DNA treatment of diabetes.