人骨髓间充质干细胞体外培养方法的改良  

An advanced method for the isolation and culture of human bone marrow mesenchymal stem cells in vitro

作　　者：叶兴德[1] 冉茜[1] 邓小军[1] 相丽欣[1] 肖燕妮[1] 李忠俊[1] 

机构地区：[1]第三军医大学新桥医院输血科,重庆400037

出　　处：《中国输血杂志》2013年第4期310-315,共6页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金(81102080)

摘　　要：目的改良人骨髓间充质干细胞(HBMSCs)体外分离培养的方法。方法采用含体积比为10%胎牛血清的α-MEM培养体系,通过密度梯度离心和贴壁筛选法从人骨髓中分离培养贴壁细胞,流式检测该细胞表面标志物,体外诱导成脂、成骨、成软骨分化并鉴定该细胞的多项分化能力,集落克隆形成和MTT试验检测该细胞的活力和增殖能力,并分别与经典的Dexter长期培养体系比较。结果采用改良方法从人骨髓中分离、培养出具有塑料底物粘附性的细胞,目的细胞不表达或低表达CD14、CD34、CD45,高表达CD73、CD90、CD105;体外成功诱导脂肪细胞、骨细胞、软骨细胞分化;Dexter-LTC体系培养的集落个数(为8.0±1.0)个、单个集落细胞数为(59.2±8.2)个,改良法培养的集落个数为(11.3±1.53)个、单个集落中细胞数为(66.5±14.4)个(P<0.05);在Dexter-LTC培养体系下,BMSCs于培养d10进入对数生长期、d19进入平台期,改良法培养BMSCs d4进入对数生长期、d15进入平台期。结论成功建立了改良的HBMSCs体外分离培养体系,且该方法培养的HBMSCs体外扩增速度、增殖能力、克隆形成能力明显改善。Objective To improve the method for tile isolation and culture of bone marrow mesenchymal stem cells in vitro. Methods Using the culturing system of c^-MEM containing 10% FBS, the adherent ceils were isolated and cultured from human bone marrow by density gradient centrifugation and adherent cells filter method. The surface markers of these cells were detected with flow eytometry. The adherent cells were directed induced and differentiated into osteoblasts, adipo- cytes and chondroblasts in vitro to indentify the muhipotent ability. The vitality and regeneration ability of these ceils were detected using colony forming unity assay and MTF assay ,then compared the vitality and regeneration ability of the cells u- sing the new method to the cells using the Dexter-LTC method respectively. Results The ceils isolated and cultured from human bone marrow using our system presented adherent in the bottom of the plastics material. Flow cytometry showed that they had low or no expression of CD14 ,CD34 and CD45 ,while they had high expression of CD73, CD90 and CD105. They could be induced to differentiate into osteoblasts, adipocytes and chondroblasts in vitro. With the Dexter-LTC method, the colony unit number was 8.0 -＋ 1.0, and the number of cells per colony was 59. 2 ＋ 8. 2. With the new method,the colony u- nit number was 11.3 - 1.53, and the number of cells per colony was 66. 5 _＋ 14. 4. The elonogenic ability of MSCs between them differed significantly（P 〈 0. 05 ）. MSCs with the Dexter-LTC method were in Logarithmic growth period in the 10th day, and in platform period in the 19th day. While MSCs with the new method were in Logarithmic growth period in the 4th day,and in platform period in the 15th day. Conclusion An advanced system for tbe culture of bone marrow mesenchymal stem ceils in vitro was successfully established. And with the new method ,the proliferation speed, proliferation ability, the colony forming unity ability were improved obviously.

关 键 词：骨髓 间充质干细胞 体外培养 诱导 细胞分化 集落 

分 类 号：R457.7[医药卫生—治疗学] R329.26[医药卫生—临床医学]