慢病毒介导CX43基因修饰小鼠骨髓间充质干细胞体系的建立及鉴定

Construction and identification of lentivirus mediated CX43 gene modified mouse bone marrow mesenchymal stem cells

目的建立慢病毒介导间隙连接蛋白43(CX43)基因修饰小鼠骨髓间充质干细胞(BMSCs)体系并进行鉴定,为研究CX43过表达BMSCs在移植物抗宿主病中的作用奠定实验基础。方法采用全骨髓贴壁分离培养法获取小鼠BMSCs,流式细胞术检测细胞表面标志和成骨、成脂、成内皮细胞诱导分化能力;重组慢病毒载体pHBLV^(TM)-CX43-GFP、pHBLV^(TM)-GFP转染BMSCs,未转染慢病毒的BMSCs为空白对照,应用细胞免疫荧光、免疫组化法和Western blot方法检测各组CX43的表达。结果流式细胞术检查示BMSCs表达CD73,CD44,CD29和CD90,不表达CD34,CD105;成骨诱导21d茜素红染色呈红色结节,成脂诱导分化14d可见明显脂滴并油红染色阳性,成内皮细胞诱导分化21d免疫荧光示内皮细胞标识CD31,CD34表达明显增加,接种于Matrigel基底胶上具有成小管能力;免疫荧光、免疫组化和Western blot方法均显示CX43基因修饰的BMSCs的CX43蛋白表达明显增加。结论成功建立了小鼠BMSCs分离培养法和CX43基因体外转染BMSCs体系。

Objective To construct and identify the method of lentivirus mediated CX43 gene modified mouse bone marrow mesenchymal stem cells（BMSCs） in vitro.Our aim is to investigate the effect of BMSCs transfected with pHBLV^TM-CX43-GFP in treatment of graft versus host disease.Methods BMSCs was adopted by whole bone marrow adherence method,the specific surface markers was defected,osteogenic,adipogenic induction and endothelial cells differentiated were performed on BMSCs.Then,CX43 protein was detected by immunofluorescence,histochemistry and Western blot in BMSCs and BMSCs transfected with pHBLV^TM-CX43-GFP or pHBLV^（TM）-CX43-GFP.Results Flow cytometry showed that BMSCs were positive for CD73,CD44,CD29,CD90,and negative for CD34,CD105.After 21 days of osteogenic induction,alizarin red stain showed that alizarin red was positive in cells,After 14 days of adipogenic induction,lipid droplets of cells was obviously discovered and oil red staining positive.The result of BMSCs differentiation into endothelial cells showed that CD31 and CD34 expressions increased significantly by immunofluorescence after 21 days of induction,and these cells could form capillaries tube-like structure on Matrigel.Immunofluorescence,immunohistochemistry and Western blot methods confirmed that the level of CX43 protein was significant increased in BMSCs transfected with pHBLV^TM-CX43-GFP.Conclusions We have successfully established a method of murine BMSC isolated culture and the methods of pHBLV^（TM）-CX43-GFP transduced into BMSCs.