转胰岛素基因的人骨髓间充质干细胞治疗大鼠糖尿病

Application of Human Mesenchymal Stem Cells Transfected with Insulin Gene Treatment of Diabetes of Diabetic Rat

目的通过基因转染构建分泌胰岛素的人骨髓间充质干细胞(IPhMSCs);移植IPhMSCs治疗大鼠糖尿病。方法将人胰岛素基因插入逆转录病毒载体pLNCX的多克隆位点中,构建重组表达载体pLNCX/hIns,经包装细胞PT67包装后转染人骨髓间充质干细胞(hMSCs),经G418筛选出阳性克隆后培养,获得IPhMSCs。将IPhMSCs移植到链脲佐菌素(STZ)诱导的糖尿病大鼠肾包膜下观察疗效。结果重组表达载体pLNCX/hIns转染hMSCs后,人胰岛素基因在hMSCs中稳定表达3周以上,但IPhMSCs体外葡萄糖刺激释放试验阴性。糖尿病大鼠在移植IPhMSCs后,血糖水平下降(P<0.05),体内胰岛素水平上升,体质量增加,糖尿病症状改善,生存时间延长,腹腔糖耐量试验(IPGTT)接近正常水平。结论通过基因工程技术可使外源性胰岛素基因在hMSCs中有效表达;构建的IPhMSCs能够降低糖尿病大鼠血糖,改善症状,延长生存时间。

Objective To generate human mesenchymal stem cells（hMSCs） transfected with the recombinant retroviral vectors pLNCX/hlns encoding the human insulin gene in vitro. To investigate application of transplantation of transgenic insulin-producing hMSCs （IPhMSCs） in treatment of treated diabetes of streptozotocin（STZ）-induced diabetic SD rat. Methods The human insulin cDNA was inserted into pLNCX to construct the recombinant retroviral vectors. The resulting plasmid, pLNCX/hIns, was transfected into the retroviral packaging cell line PT67. Then the retroviral supernatant was transfected in hMSCs in vitro. The successfully transfected positive clones were selected with antibiotics G418. About 5×10^6-1×10^7 transgenic hMSCs which produce insulin for more than 3 weeks in vitro were transplanted under the right renal subcapsule of STZ-induced diabetic SD rat. Blood glucose level, insulin content, weight and survival ratio was monitored regularly. To determine whether blood glucose level would be regulated in IPhMSCs transplanted rats, intraperitoneal glucose tolerance test（IPGTT） was performed four weeks after transplantation. Results The recombinant retroviral vectors pLNCX/hIns carrying insulin gene were constructed successfully, hMSCs transfected with the recombinant retroviral vectors successfully expressed human insulin gene and synthesized human insulin in vitro. Transplantation of insulin-producing hMSCs reduced blood glucose （P〈0.05）, raised insulin content, alleviated diabetic symptom and prolonged survival of diabetic SD rat immunosuppressed with cyclosporine A. Conclusion It suggests that this transgenic hMSCs with external insulin gene can produce insulin in vitro and control diabetes of diabetic rat effectively.