绿色荧光蛋白基因逆转录病毒载体转染人骨髓间充质干细胞及表达的研究

目的　绿色荧光蛋白 (EGFP)基因的逆转录病毒载体转染人骨髓间充质干细胞(MSCs)及其表达情况。方法　pEGFP与逆转录病毒载体经过酶切、连接等构建重组的EGFP 逆转录病毒载体 ,转染PA3 17细胞 ,用G418筛选出抗性克隆 ,获病毒上清 (滴度达到 8.5× 10 5cfu/ml)并感染人MSCs。流式细胞仪测定转染效率后加入成骨细胞分化培养夜 (地塞米松 (10 -7mol/L)、β 甘油磷酸钠 (10mmol/L)、维生素C(5 0mg/L ) ) ,2周后观察转染细胞的分化情况。 结果　 48h后细胞转染效率为 7% ,G418筛选 2周后约 97%细胞出现抗性克隆 ,表达维持 4周。基因转染细胞能够分化为成骨细胞。结论　重组EGFP逆转录病毒载体能转染MSCs ,且不影响细胞的功能。

Transfection of enhenced green fluorescent protein gene into mesenchymal stem cells and its expression

Objective As amarker gene,Enhanced green fluorescent protein(EGFP) was widely applied to the investigation of scope of gene therapy.A variety of studies have demonstrated that Mesenchymal stem cells (MSCs) may be an iedal target cell type in the gene therapy.Methods To investigate the possibility,MSCs were transfectant by a retrovirus vector carrying the EGFP gene.Results Flow cytometry analysis showed that 7% of the cells were positive for EGFP and the percentage increased up to 97% after the cells were screened by neomycin.Moreover,the transfected cells still possessed the capacity of differentiation into osteoblasts,as shown by alkaline phosphatase activity.Conclusion The result shows a highlight on the clinical applicability of MSCs in gene therapy.