大鼠骨髓间充质干细胞的分离培养与初步鉴定

目的探讨体外分离、传代培养大鼠骨髓问充质干细胞（mesenchymal stem cells，MSCs）的方法，并对培养细胞进行初步鉴定，为利用大鼠骨髓MSCs进行感音神经性聋细胞移植治疗的研究奠定基础。方法用贴壁培养法分离大鼠骨髓细胞，第1组于24小时全量换液，第2组于24小时半量换液，第3组于3天全量换液，传代扩增。相差显微镜进行形态学观察；RT—PCR检测培养细胞表面分子表达；定向诱导培养细胞向成脂细胞、成骨细胞方向分化。结果24小时首次全量换液组培养7天后观察见少量细胞集落，细胞形态不规则；24小时首次半量换液组培养7天后观察见较多细胞集落，细胞规则，呈梭形、椭圆形；3天首次全量换液组培养7天后观察见较多悬浮细胞．与贴壁细胞混杂在一起。培养细胞表面分子SH2、CD31、CD44呈阳性表达，但不表达CD34。第2组培养细胞传代后加入不同诱导剂定向诱导分化一定阶段，分别经油红O及VOFIKossa法染色鉴定证实MSCs可分别向脂肪细胞及成骨方向分化。结论本实验分离培养的大鼠骨髓原代细胞24小时首次半量换液培养利于大鼠骨髓MSCs的分离和纯化，可稳定表达SH2、CD31、CD44。MSCs具有多项分化潜能。

Isolation,cultivation and preliminary identification of rat bone marrow mesenchymal stem cells

Objective To establish a method of isolating and culturing rat bone marrow mesenchymal stem cells (MSCs) in vitro and to analyse their phenotypical properties after culture expansion as well as preliminary identification,so as to establish foundation for the celluar transplant therapy of rat bone marrow MSCs in sensorineural hearing loss.Methods Rat bone marrow cells were separated and expanded by adherence culture.The medium of the first group was all changed after 24 hours.The second group was half changed after 24 hours.The third was all changed after 3 days.The morphology of cells was observed with phase-contrast microscope.The surface molecule expressions of cells was examined by RT-PCR.After expanded in α-MEM supplemented with 10% FBS and passaged,the muhilineage differentiation capability of cells was examined by culturing cells under conditions favorable for adipogenic and osteogenic differentiation in vitro.Results In the first group that the medium was firstly all changed after primary culture for 24 hours,few cell colonies adhering to culture plastic that cells exhibited irregular shape were observed.When,the medium was firstly half changed after 24 hours,a few cell colonies were obtained,with fusiform and oval shape.In the third group,a few suspension cells,mixed with adhered cells,were difficult to remove.After passaged,RT-PCR indicated that rat bone marrow MSCs expressed SH2,CD31,CD44,and lacked expression of CD34.By culturing in adipogenic medium for 8 days and in osteogenic medium for 4 weeks respectively,and then by identified by Oil Red O and yon Kossa staining,the cells of passage 3 were successfully induced to adipocytes and osteogenesis.Conclusion The method that medium is firstly half changed after primary culture for 24 hours is benefit for isolation and purity of rat MSCs.The cells isolated in this experiment have biolobical characteristics of adherence to plastic and specific surface antigen(Ag) expression of SH2,CD31,CD44,which sugge that it can be as muhipotent differentiation potential.