小鼠骨髓间充质干细胞的体外分离培养和鉴定方法学探讨  

To discuss the way for isolation,culture and identification of mice bone marrow mesenchymal stem cells in vitro

作　　者：银广悦[1] 陈素萍[2] 丁俊丽[1] 张继领[1] 刘继勇[1] 张龙[1] 张明[1] 

机构地区：[1]中国石油天然气集团公司中心医院检验科,河北廊坊065000 [2]中国石油天然气集团公司中心医院病理科,河北廊坊065000

出　　处：《中国实验诊断学》2013年第4期647-650,共4页Chinese Journal of Laboratory Diagnosis

基　　金：廊坊科技支撑项目(2007050257)

摘　　要：目的探讨骨髓间充质干细胞体外分离培养和鉴定方法,找到一种能在质和量上均能满足需要的体外培养条件。方法无菌条件下分离处死后取小鼠双下肢骨,低糖DMEM培养液冲出骨髓,离心5mins,用5ml含10%胎牛血清的低糖DMEM培养液重悬细胞。加入等体积密度为1.073g/L percoll分离液离心30mins。取中层云雾状细胞。37℃、5%CO2培养箱中贴壁培养。取5-10代细胞培养至对数生长期后,洗涤离心。取100μl细胞悬液分别加入相应抗体,避光孵育30mins,洗涤离心。流式细胞仪检测,CELLQUEST软件收集数据。结果骨髓间充质干细胞是一类纺锤形或梭形细胞,与纤维细胞类似,顺向呈"鱼群样"排列。CD45、CD34、CD31呈阴性表达,表达率分别为0.82%,0.27%,0.97%;CD44、CD90、CD71呈阳性表达,表达率分别为99.8%,99.7%,99.36%。不同代数细胞表面分子的表达情况无明显差异(P>0.05)。结论贴壁培养法能够在质和量上满足骨髓间充质干细胞的体外培养条件。Objective To investigate the＇methods which isolate,culture and identify bone marrow mesenchymal stem cells in vitro, find a better cell culture condition to satisfy their quality and quantity. Methods The mice were killed un- der sterile conditions,both lower extremities wePe removed,and bone marrows are washed out by DMEM medium with lower carbohydrate,centrifuged for 5 min, resuspended in 5 ml DMEM medium containing 10 % FBS. Then added equal volume percell separation liquid which density is 1. 073 g/L,centrifuged for 20 rain,the middle cloudy cells were isola- ted. The cells were grown adhering the plates in DMEM supplemented with 10% FBS, at 37 ~C in a humidified atmos- phere of 5% CO2. when the cells which wereqn 5-10 generations were cultured in exponential growth phase,the cells were digested,washed and centrifuged, Cell suspension （100 /al） were treated with antigen-specific monoclonal antibod- ies and incubated for 30 ＂min in dark condition, then washed and centrifuged. Flow cytometry was carried out using a CELLQUEST acquisition and analysis software. Results with similar to the fibroblasts, Bone marrow mesenchymal stem cells are a class of fusiform or spindle-shape cells,which arrange in same direction,like school of fish,and CD45, CD34,CD31 are negative expression, the rate of expression are 0. 82% ,0. 27% ,0. 970/00, respectively~ CD44,CD90, CD71 are positive expression,the rate of expression are 99.8%, 99.7%, 99.36%, respectively. Conclusion Culturing bone marrow mesenchymal stem cells in adherence＇ method can ensure tire quality and quantity of the cells in vitro.

关 键 词：贴壁法 骨髓间充质干细胞 体外培养 鉴定 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]