大鼠骨髓间充质干细胞的培养与鉴定

Culture and identification of rat bone marrow mesenchymal stem cells

背景:骨髓间充质干细胞在骨髓中含量极低,体外培养难度较大。体外分离培养纯度高、活力强、生物特性均一的间充质干细胞,对组织工程及细胞的体内、体外实验显得至关重要。目的:建立大鼠骨髓间充质干细胞的分离、培养、纯化方法,并进行细胞形态学观察、表面标志物鉴定及多向分化能力检测。方法:通过全骨髓贴壁法体外分离、培养、纯化大鼠骨髓间充质干细胞,进行形态学观察,绘制生长曲线,细胞周期分析,流式细胞仪检测细胞表面标记物,分别向成骨、成脂方向诱导分化。结果与结论:大鼠骨髓间充质干细胞生长以梭形细胞为主,呈放射状排列的细胞集落,细胞生长旺盛,可连续稳定传代10代以上。生长曲线及细胞周期显示骨髓间充质干细胞符合正常细胞生长特征且生长活跃。第3代骨髓间充质干细胞CD44,CD90,CD105均呈阳性表达,而CD34,CD45呈阴性表达。成脂、成骨诱导后,油红O染色、碱性磷酸酶染色、von Kossa法染色和茜素红染色均呈阳性。全骨髓贴壁培养法操作简单,可大量分离、纯化、扩增骨髓间充质干细胞,所获细胞具有间充质干细胞的一般生物学特性,经诱导培养后具有多向分化潜能。实验所用的全骨髓贴壁法法为组织工程提供充足的种子细胞来源具有重要的现实意义。

BACKGROUND：Bone marrow mesenchymal stem cells（BMSCs） are not rich in bone marrow,and are difficult to culture in vitro.It is crucial to tissue engineering and to the cell experiments in vivo or in vitro that isolating and culturing in vitro BMSCs with high purity,strong vitality and homogeneous biological characteristics.OBJECTIVE：To establish a method of isolation,cultivation and purification of rat BMSCs in vitro,to observe cell morphology,and to assess surface markers and multi-directional differentiation capacity.METHODS：BMSCs from rats were isolated,cultured and purified by the whole bone marrow adherence method,for morphology observations,the growth curve was drawn,cell cycle was analyzed,cell surface markers were assessed by flow cytometry,and BMSCs were induced to differentiate into osteoblasts and adipocytes.RESULTS AND CONCLUSION：BMSCs from rats were spindle cell-based,showing radial colony arrangement.Cells kept strong growth and could passage in continuous and stable manner over 10 passages.The growth curve and cell cycle demonstrated that BMSCs were consistent with the growth characteristics and good activity of normal cells.At the third passage,BMSCs were negative for CD34 and CD45,but positive for CD44,CD90 and CD105.Following induction,oil red O staining,alkaline phosphatase staining,von Kossa staining and alizarin red staining produced a strong reaction in cells.Whole bone marrow adherence method is simple and can isolate,purify and amplify BMSCs in vitro.The obtained cells have general biological characteristics of mesenchymal stem cells,and also have potentiality of multi-directional differentiation.This experimental method has important practical significance to provide adequate source of seed cells for tissue engineering.