建立大鼠骨髓间充质干细胞稳定分离培养体系与鉴定  

Establishment of a stable isolation culture system and identification of rat bone marrow mesenchymal stem cells

作　　者：杨丽[1] 张荣华[1] 谢厚杰 蔡宇[1] 黄丰[3] 

机构地区：[1]暨南大学药学院中药学教研室,广东省广州市510632 [2]广东省荣誉军人康复医院医务科,广东省广州市510260 [3]暨南大学药学院中药药理学教研室,广东省广州市510632

出　　处：《中国组织工程研究与临床康复》2009年第6期1064-1068,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30472274);国家中医药管理局中医药科学研究专项(04-05JL19);广东省自然科学基金重点项目(04105837);广东省社会发展计划重点引导项目(2004B33001004)~~

摘　　要：背景:骨髓间充质干细胞作为种子细胞在组织工程等领域应用广泛,建立稳定的分离培养体系和统一鉴定标准尤为重要。目的:以不同方法体外分离培养大鼠骨髓间充质干细胞,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。设计、时间及地点:细胞学体外对比观察,于2005-08/12在暨南大学完成。材料:SPF级3月龄SD雌性大鼠5只,由中山大学实验动物中心提供。方法:无菌条件下分离大鼠双下肢,低糖DMEM培养液冲出骨髓,分别运用全骨髓贴壁法和密度梯度离心法体外分离骨髓间充质干细胞,利用差速贴壁原理对细胞进行纯化扩增。取生长状态良好的第3代骨髓间充质干细胞,分别加入成骨、成脂、成软骨诱导培养基,培养14d。主要观察指标:倒置相差显微镜下观察骨髓间充质干细胞的生长状态,流式细胞仪检测细胞表面标志物的表达,采用碱性磷酸酶染色鉴定成骨能力,以油红O染色鉴定成脂能力,以甲苯胺蓝染色鉴定成软骨能力。结果:与密度梯度离心法相比,全骨髓贴壁法分离获得的骨髓间充质干细胞贴壁时间短,增殖快,经传代后能够纯化。第3代骨髓间充质干细胞形态单一均匀,呈典型的极性漩涡状生长,不表达造血前体细胞标志抗原CD34和白细胞标志抗原CD45,表达整合素家族成员CD29和黏附分子CD44,经诱导后碱性磷酸酶染色、油红O染色和甲苯胺蓝染色均呈阳性。结论:采用全骨髓贴壁法可稳定获得均质性良好的大鼠骨髓间充质干细胞,效果优于密度梯度离心法。经全骨髓贴壁法体外分离培养的细胞在形态学、细胞表面标志物表达和多向分化能力方面具有干细胞生物学特性,经鉴定为大鼠骨髓间充质干细胞。BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） are widely used as seed cells in tissue engineering and other fields. Therefore, the establishment of a stable isolation and identification system of BMSCs is very important. OBJECTIVE： To in vitro harvest rat BMSCs through various methods, to observe cell morphology, and to assess surface marker and multi-directional differentiation. DESIGN, TIME AND SETTING： This cytology in vitro controlled experiment was performed at the Jinan University from August to December 2005. MATERIALS： Five SPF grade female Sprague Dawley rats, aged 3-months old, were provided by the Animal Experimental Center of Sun Yat-sen University. METHODS： Rat double lower limbs were isolated sterilely. Bone marrow was obtained by rinsing using low-glucose DMEM. BMSCs were isolated and purified by the whole bone marrow adherence method and density gradient centrifugation. BMSCs were purified by the differential adherence. At the third passage, BMSCs were incubated in osteogenous, adipogenic and chondrogenic medium for 14 days. MAIN OUTCOME MEASURES： The morphology of BMSCs was observed by the inverted phase contrast microscope. Expression of BMSC surface antigens was detected by flow cytometer. Osteogenous potential was assessed by alkaline phosphatase staining Adipogenic potential was evaluated by Oil red O staining. Chondrogenic potential was assessed by toluidine blue staining. RESULTS： Compared with the density gradient centrifugation, BMSCs isolated by the adherence separation had shorter adherence time and faster multiplication rate, which could be purified by subculture. At the third passage, BMSCs showed the typical polar swirl morphology. BMSCs were negative for CD34 and CD45, but positive for CD29 and CD44. Following induction, alkaline phosphatase staining, Oil red staining and toluidine blue staining produced a strong reaction in cells. CONCLUSION： BMSCs by the adherence separation method have a good homogeneity, and the outcome is better compared with the de

关 键 词：骨髓间充质干细胞 大鼠 分离 鉴定 

分 类 号：R394.2[医药卫生—医学遗传学]