胰岛素基因逆转录病毒载体在人骨髓间充质干细胞表达的实验研究

Construction of Retroviral Vector Carring Insulin Gene and Its Stable Expression in Bone-marrowed Mesenchymal Stem Cells

目的构建携带胰岛素基因逆转录病毒载体,使其在人骨髓间充质干细胞(hMSCs)内稳定表达。方法通过RT-PCR法从人胰腺组织中提取胰岛素基因片段,并将其连接入逆转录病载体pLNCX,经包装细胞PT67的包装后,获得含外源性胰岛素基因的病毒颗粒(病毒滴度达4.2×106cfu/ml)并感染hMSCs,经过G418筛选后,用RT-PCR法、免疫荧光法、放射免疫法、葡萄糖刺激试验分析胰岛基因的表达情况。结果携带胰岛素基因的逆转录病毒载体构建成功,转导入hMSCs后能够稳定表达胰岛素基因,并分泌至细胞外持续3周以上,但对葡萄糖刺激无明显反应。结论重组人胰岛素基因逆转病毒载体能够将胰岛素基因导入hMSCs并稳定表达。

Objective To construct retroviral vector carting human insulin gene and investigate its expression in human bone-marrowed mesenchymal stem cells （hMSCs）. Methods The recombinant retroviral vector INS-pLNCX was constructed by cloning the human insulin cDNA into the retroviral vector pLNCX containing multiple cloning site. The INS-pLNCX vector was transfected into ecotropic packaging cell line PT67 by clonfectin method. Then the supernatant of PT67 was used to infect hMSCs. RT-PCR and Immunofluoresence was used to test the expression of insulin gene in hMSCS. Result The recombinant retroviral vector carring insulin gene was successfully constructed. By transfecting PT67 cell, recombinant retrovirus with infective capability was packaged and used to infect hMSCs. The infected hMSCs can stably express human insulin more than 3 weeks, but can＇t react to high level of glucose. Conclusion The hMSCs infected by recombinant retroviral vector carting insulin gene can provide a new way to deal with the shortage of β-cell for transplantation.