全骨髓贴壁法分离培养大鼠骨髓间充质干细胞及其诱导分化

Isolation,culture and multi-directional differentiation of rat bone marrow mesenchymal stem cells by using the whole bone marrow adherence method

背景:骨髓中的间充质干细胞含量极少,每1×104～1×105个单核细胞中约有1个骨髓间充质干细胞,需要在体外分离、纯化、扩增后才能满足体内移植要求。目的:观察体外分离培养的骨髓间充质干细胞生物学特性及多向分化潜能。设计、时间及地点:细胞学体外观察,于2007-10/2008-12在福建医科大学附属协和医院泌尿外科研究室完成。材料:清洁级雄性近交系SD大鼠30只,由上海斯莱克实验动物有限公司提供。方法:通过全骨髓贴壁法体外分离培养大鼠骨髓间充质干细胞,待细胞融合至80%～90%胰蛋白酶消化传代。取传至第3代细胞,分别向成骨、成脂方向诱导分化。主要观察指标:倒置相差显微镜下观察细胞形态,流式细胞仪检测其表面标记,茜素红染色检测其成骨能力,油红O染色检测其成脂能力。结果:原代及传代的骨髓间充质干细胞均呈长梭形成纤维细胞样,连续传代10代以上,细胞始终保持旺盛的生长及扩增能力。第3代骨髓间充质干细胞CD90,CD44,CD106均呈阳性表达,而CD34,CD45,CD11b呈阴性。成骨诱导21d后,可见大量橘红色矿化结节形成。成脂诱导2周后,可见细胞的胞浆内出现大量红染脂滴。结论:全骨髓贴壁培养法可大量分离纯化、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。

BACKGROUND： There is few mesenchymal stem cells （MSCs） in the bone marrow, about one BMSC in 1×10^4～1 × 10^5 monocytes Following in vitro isolation, purification and amplification, it is satisfactory for in vivo requirement. OBJECTIVE： To observe biological characteristics and potentiality of multi-directional differentiation of BMSCs in vitro. DESIGN, TIME AND SETTING： The cytological in vitro study was performed at the Institute of Urinary Surgery, Union Hospital Affiliated to Fujian Medical University from October 2007 to December 2008. MATERIALS： A total of 30 clean male Sprague Dawley rats of inbreeding line were supplied by Silaike, Shanghai, China. METHODS： BMSCs from rats were isolated and cultured in vitro by using the whole bone marrow adherence method. When 80%-90% confiuency, BMSCs were digested by trypsin. At the third passage, BMSCs were induced to differentiate into osteoblasts and adipocytes. MAIN OUTCOME MEASURES： Cell morphology was observed under the inverted phase contrast microscope. BMSCs surface markers were detected using flow cytometry. Osteogenic ability was examined by alizarin red staining. Adipogenic ability was measured by Oil red O staining. RESULTS： The primary cells and the passage cells were mostly fusiform in shape, to be similar to fibroblasts. Cell still kept a high potential of growth and amplification following over 10 subcultures. At the third passage, BMSCs were positive for CD44, CD90, CD106, but negative for CD34, CD45, CD11 b. Following 21 days of osteogenic induction, cell alizarin red staining showed that alizarin red was positive in osteoblasts. Following 2 weeks of adipogenic induction, oil red O staining showed that red lipid droplet existed in adipocytes. CONCLUSION： Whole bone marrow adherence method can isolate, purify and amplify BMSCs in vitro. The obtained cells have general biological characteristics of MSCs, and also have potentiality of multi-directional differentiation.