人脐带间充质干细胞的体外获取、鉴定及标记

Obtaining,Identifying and Marking of the Human Umbilical Cord-derived Mesenchymal Stem Cellsin Vitro

目的从人脐带中获取间充质干细胞(MSC)并进行标记,为进一步移植治疗奠定基础。方法运用组织块贴壁法从人脐带中分离MSC,通过传代培养后,进行形态学观察;采用流式细胞仪对人脐带间充质干细胞(h UC-MSC)的细胞表型进行鉴定;通过特异性染色鉴定体外成脂、成骨诱导后的分化能力;运用免疫荧光证实Brd U标记细胞是否成功。结果运用组织块贴壁法可成功从人脐带中分离出MSC,培养1周左右可见细胞从组织块边缘爬出,向外增殖生长,形成集落;流式细胞仪检测P5代细胞,高表达CD73、CD90及CD105,不表达或低表达CD14、CD34、CD45、CD79a及人类白细胞抗原-DR;成脂诱导2周,油红O染色为阳性,成骨诱导3周,茜素红染色出现红色沉淀物;Brd U标记细胞经免疫荧光检测后,可见红色荧光。结论运用组织块贴壁法从人脐带中分离的细胞,具备MSC的特点并可成功被标记,可用于细胞移植领域的研究。

Objective To obtain the mesenchymal stem cells（MSCs） from human umbilical cord and mark in vitro, for further transplantation therapy. Methods The MSC were isolated from human umbilical cord by tissue explants culture method. After subculture in vitro, the morphology of h UC-MSC was observed; the surface antigens of h UC-MSC were detected by flow cytometry; adipogenic and osteogenic differentiation were determined by specific staining; h UC-MSC labelled with Brd U were identified by immunofluorescence. Results MSC could be isolated successfully by tissue explants culture method. When cultured about one week, the cells climbed out from the tissue block edge, proliferated and formed colonies; the h UC-MSCs of passage 5 were detected by flow cytometry, and they highly expressed CD73, CD90 and CD105, didn＇t express or lowly expressed CD14, CD34, CD45, CD79 a and human leukocyte antigen-DR. After two weeks of adipogenic induction, they were positive in oil red O staining, and after three weeks of osteogenic induction, red precipitate could be seen by alizarin red staining, and the red fluorescence of the h UCMSC labelled with Brd U could be detected by immunofluorescence detection. Conclusion The cells can be isolated from human umbilical cord by tissue explants culture method, with the characteristics of h UC-MSCs and can be labeled successfully in vitro, so it can be used for the research in the field of cell transplantation.