绿色荧光蛋白基因转染大鼠骨髓间充质干细胞的实验研究

A study on transfecting green fluorescent protein gene to rat bone marrow mesenchymal stem cells

目的 体外研究绿色荧光蛋白 (GFP)基因转染骨髓间充质干细胞 (MSCs)的方法,并检测基因转染后细胞的生物学特性及分化潜能。方法 体外分离培养大鼠MSCs,用重组GFP的腺病毒载体Ad GFP及脂质体介导质粒pEGFP C1两种方法转染GFP,流式细胞术观察比较基因转染和表达结果;将腺病毒介导的基因转染细胞经成骨定向诱导后,检测碱性磷酸酶表达和钙结节形成情况。结果 重组Ad GFP对细胞的感染率达(41 3±1 4)%,感染 6周后仍有表达;脂质体组转染效率最高为 12 5%。病毒感染组阳性细胞与未感染组细胞经成骨诱导分化后碱性磷酸酶表达均逐渐增高;诱导 3周后茜素红钙盐染色均为阳性。结论 重组GFP的腺病毒载体Ad GFP可高效感染MSCs,基因转染后细胞的增殖分化能力与未转染细胞差异无统计学意义(P>0 05),可以作为一种高效的大鼠MSCs的标记方法。

Objective To study an efficient method to transfect green fluorescent protein gene (GFP) to rat bone marrow mesenchymal stem cells (MSCs) and to dertermine the biological properties and differentiation potency of transfected MSCs.Methods SD rats′ bone marrow MSCs were separated and purified in vitro. After subculture and expansion, MSCs infected with Adenoviral vector (Ad GFP) or transfected with liposome were observed, and their transfection efficiency was assessed with flow cytometry. The MSCs expressing GFP gene were induced to differentiate to osteoblast, and non transfected MSCs were set as control. Results Ad GFP delivered GFP gene with high efficiency to rat MSCs. (41 3±1 4) % of MSCs infected with Ad GFP expressed GFP gene, which was much higher than the control (12 5%). Expression of GFP gene of infected MSCs maintained stable from 1 to 6 weeks after infection. Infected MSCs possessed the same alkaline phosphatase activation as non infected MSCs, and formed mineralized mouldes.Conclusions The infected MSCs with Ad GFP expressed GFP with much higher efficiency than liposome transfection, and maintained the same ability of proliferation and differentiation as non infected MSCs. Transgection with Ad GFP is a highly effective method for labeling MSCs.