兔骨髓间充质干细胞的分离、体外培养、鉴定和体外标记  

Isolation,culture,identification and labeling of bone marrow-derived mesenchymal stem cell of rabbit in vitro

作　　者：朱峰[1] 胡贞贞[2] 郭光华[3] 王红梅[2] 

机构地区：[1]南昌大学第一附属医院重症医学科,南昌330006 [2]南昌大学医学院病理生理学教研室 [3]南昌大学第一附属医院烧伤科

出　　处：《南通大学学报（医学版）》2010年第3期157-160,163,F0003,共6页Journal of Nantong University(Medical sciences)

基　　金：江西省教育厅青年科学基金项目(GJJ09432)

摘　　要：目的:探讨分离、体外培养、鉴定及体外标记兔骨髓间充质干细胞(bone marrow-derived mesenchymal stemcells,MSCs)的方法,为进一步的实验研究打下基础。方法:选择健康幼龄新西兰大耳白兔,于双侧髂后上嵴及胫骨上端内侧部行骨髓穿刺提取骨髓。采用全骨髓培养法(直接贴壁法)分离培养MSCs,对第2、3、4、5、6代MSCs应用MTT法测定生长曲线,分析MSCs的生长规律。对MSCs进行形态学观察,通过流式细胞术对CD34、CD44、CD45、CD105这4种MSCs表面抗原进行鉴定,证明所培养的细胞为MSCs。采用5-溴脱氧尿嘧啶核苷(5-Bromo-2-deoxyuridine,BrdU)体外标记兔MSCs,检测其标记阳性率。结果:全骨髓培养法培养的原代MSCs接种4天后可以被观察到,形态均匀成梭形,生长增殖迅速,符合MSCs生长的特性,7～8d MSCs融合接近80%,传代培养生长良好。MSCs生长曲线呈S型,由生长曲线分析可知,MSCs在培养第4～8d为高速生长期,MSCs在第3～5代生长最为旺盛。经流式细胞术鉴定,CD34(-)、CD45(-)、CD44(+)、CD105(+),证明所培养的细胞为较纯的MSCs。BrdU体外标记兔MSCs的阳性率达到85%～90%。结论:应用本实验方法,可以分离、纯化培养兔骨髓间充质干细胞且操作简便,效率高,经济实用。所培养的MSCs体外生长稳定、增殖速度快、贴壁率高、可连续传代,可用于MSCs功能及应用的进一步研究。应用BrdU体外标记兔MSCs是安全可靠的。Objective：To explore a method of isolation,purification and culture of bone marrow-derived mesenchymal stem cells（MSCs） of rabbits in vitro for the sake of further study.Methods：Bone marrow tissue was harvested from bilateral posterior iliac crest and tibias bone of young New Zealand white rabbit.MSCs were isolated and proliferated by the method of whole bone marrow culture.Growth curves of the 2nd,3rd,4th,5th and 6th generation MSCs were drawn by MTT method. CD34,CD44,CD45,CD105 antigens of MSCs were identified by flow cytometry.The purified MSCs were labelled with BrdU and immunohistochemistry was performed to calculate and evaluate the labelling rate.Results：Primary cultured MSCs started adhering to plates 12 hours after seeding and spindle-shaped MSCs were observed under microscope and growing at a rapid speed.Confluence of MSCs reached to 80%7～8 days after seeding.From the MSCs growth curve,which was S shape,it was known that MSCs grew in a rapid stage at the 4th-8th day after seeding,and MSCs of 3rd～5th generation possessed higher activity.CD34（-） ,CD45（-） ,CD44（＋） and CD105（＋） were detected by flow cytometry,which confirmed finally that the cultured cells were MSCs.The positive rate of MSCs labeled by BrdU was 85%～90%.Conclusion：In this study,simple operation,high efficiency,economy and practice were characteristic of this method,and MSCs were isolated and proliferated stably and rapidly.Cultured MSCs possessed high activity of growing and amplification and were appropriate for further research. Application of BrdU labelling for MSCs in vitro was safe and reliable.

关 键 词：骨髓间充质干细胞 全骨髓培养法 细胞培养 流式细胞术 5-溴脱氧尿嘧啶核苷 兔 

分 类 号：R563[医药卫生—呼吸系统]