体外诱导骨髓间充质干细胞向上皮样细胞分化

Rabbit stem cells from bone marrow stroma differentiate into tracheal epithelial cells in vitro

目的通过体外气-液界面共培养的方法,诱导兔骨髓间充质干细胞(BMSCs)向上皮样细胞分化。方法贴壁分离法体外培养BMSCs,pMSCV/GFP逆转录病毒转染BMSCs,使BMSCs成功表达绿色荧光蛋白。同时两步酶消化法(Pronase酶和胰酶)体外培养兔气管上皮细胞(RTEs),Cytokeratin18(CK-18)单克隆抗体鉴定。建立气-液界面培养模型,将标记有GFP的BMSCs和RTEs以一定比例混匀接种到套皿内,连续共培养14 d,CK-18免疫荧光染色鉴定分化结果。结果荧光显微镜下,pMSCV/GFP逆转录病毒转染BMSCs呈现明显的绿色荧光,同时共培养后部分标记有GFP的BMSCs经CK-18荧光染色,呈现红色荧光,说明经GFP标记的BMSCs已被成功诱导分化成气管上皮。结论体外气-液界面条件下,兔BMSCs与原代培养兔RTEs共培养可以成功诱导分化为上皮样细胞,表达上皮特异性角蛋白CK-18,为BMSCs作为组织工程化气管的种子细胞奠定实验基础。

Objective To induce stem cells from bone marrow stroma （BMSCs） to differentiate into tracheal epithelium with air-liquid interface. Methods BMSCs were separated from bone marrow with different adherent time. Retrovirus vectors with pMSCV/GFP were transferred into BMSCs and tagged green fluorescence. Meanwhile, rabbit tracheal epithelial cells （RTEs） were isolated by the method of two-step enzymes （pronase and trypsin） and identified by the cytokeratinl8 （CK-18） immunofluorescence. With co-culture of BMSCs with RTEs in a special ratio by air liquid interface, BMSCs were cultured for 14 days and testified by CK-18 monoclonal antibody. Results By using a fluorescence microscopy, GFP-tagged BMSCs partly showed distinctly red fluorescence after immunofluorescent staining of the cultured cells with a monoclonal antibody against rabbit CK-18. Conclusions With co-culture of BMSCs with RTEs in a special ratio by air liquid interface, GFP tagged BMSCs are induced successfully to differentiate into the epithelial-like cells, which express important epithelia-specific marker CK-18.