人胰岛素样生长因子-1逆转录病毒载体的构建及其在骨髓基质干细胞中的表达

Construction of Retroviral Vector Encoding Human Insulin-like Growth Factor-1 and Its Expression in Bone Marrow Stromal Cells

目的构建人胰岛素样生长因子-1的逆转录表达载体,建立逆病毒介导的IGF-1基因转移系统。方法用逆转录-多聚酶链反应(RT-PCR)从人肝细胞克隆IGF-1的基因;经DNA测序分析证实后将目的基因插入逆转录病毒载体LXSN,制备pLXSN-IGF-1表达载体;借助阳离子脂质体转染包装细胞PA317,G418筛选阳性克隆,获取病毒上清;培养人骨髓基质干细胞,用病毒上清感染骨髓基质干细胞;采用RT-PCR及Western blot法检测目的基因在靶细胞的表达。结果经酶切电泳和DNA测序表明成功构建了IGF-1逆转录病毒表达载体,转染包装细胞后可以产生IGF-1逆转录病毒,病毒感染人骨髓基质干细胞后能够表达IGF1-1重组蛋白。结论成功建立逆转录病毒载体介导的IGF-1体外表达体系,能够快速、稳定地将IGF-1基因转入骨髓基质干细胞。

Objective To construct a retroviral mediated expression system of human insulin-like growth factor-1 （IGF-1）, and determine whether bone marrow stromal cells （BMSCs） can be infected by retrovirus. Methods hIGF-lcDNA was amplified in vitro from normal human liver cells by using RT- PCR and pLXSN was cloned into plasmid vector. The recombinant vector pLXSN/IGF and control vector pLXSN were transfected into packaging cell PA317 and G418 was used to select positive colony. Hu- man BMSCs cells were infected with a high titre viral supernatant. The expression of hIGF-1 in target cells were analyzed with RT-PCR assay and Western blot. Results The retroviral vector containing IGF-1 gene was constructed successfully, amphotropic PA317 retroviral producer cells were obtained by liposome transfection and G418 seleletion. The BMSCs cells were infected by medium containing IGF-1 virus. The expression of IGF-1 in transfected BMSCs was dectected by RT-PCR and Western blot analysis. Conclusion The constructed retrovirus vectors can introduce IGF-1 into BMSC cells efficiently, which may have potential effect for gene therapy of bone repair.