犬骨髓间充质干细胞的分离、培养、纯化及鉴定  

Isolation,cultivation,purification and identification of canine bone marrow mesenchymal stem cells in vitro

作　　者：吴胜东[1] 葛建军[2] 鲍峰[1] 宫为一[1] 吴宗阳[1] 

机构地区：[1]武警安徽总队医院胸心外科,安徽合肥230022 [2]安徽医科大学第一附属医院心血管外科,安徽合肥230022

出　　处：《武警医学院学报》2009年第1期15-19,F0002,共6页Acta Academiae Medicinae CPAPF

基　　金：安徽省自然科学基金资助课题(03043707)

摘　　要：【目的】探讨分离、培养、纯化和鉴定犬骨髓间充质干细胞(mesenchymal stem cells,MSCs)方法,观察MSCs体外生长特征。【方法】自犬髂后上棘抽取骨髓约10ml,1.073g/ml的Percoll分离液梯度离心,培养皿培养、换液除去非贴壁细胞,获得MSCs,通过及时、反复传代对MSCs进行纯化和扩增培养,测定生长曲线,并进行形态学观察。流式细胞仪检测扩增后MSCs细胞周期及表面抗原特性。【结果】MSCs在含10%小牛血清的IMEM中生长性状相对稳定,1、3、5代细胞生长曲线基本一致,增殖速度快。流式细胞仪检测表明MSCs强表达干细胞标记CD44,而不表达造血干细胞特异抗原CD34、CD11a、CD14和HLA-DR。第2代(P2)末MSCs周期显示有82.4%的细胞处于G0/G1期。【结论】密度梯度离心结合贴壁培养法能有效分离、纯化和培养犬骨髓MSCs。[ Objective] To explore a new method for the isolation, cultivaton, purification and identification of MSCs and observe the biological features of canine MSCs in vitro. [Methods] 10 ml bone marrow was extracted from the iliac of canine. The marrow liquid were isolated with 1.073 g/ml Percoll. MSCs were obtained by removing the non-adherent cells. Then the MSCs were purified and expanded through passage in time. The growth curve was drawn and the morphology was observed under phase contrast microscope. Cell cycle and the antigen expression of MSCs were measured with FACS. [Results] The MSCs growth behavior was quite stable in IMEM containing 100 ml/L newbem bovine serum, and the growth curves of passage 1, 3 and 5 were quite similar, exhibiting a large expansive potential. The FACS results showed that MSCs expressed antigen CD44, the symbol of stem cell, but did not express CD34, CD14 and HLA-DR. The cell cycle analysis showed that 82.4% of the passage 2 MSCs were in G0/G1 plate. [ Conclusions] The marrow MSCs could be effectively isolated, purified and expanded by density gradient centrifugation and adhering to the culture plate.

关 键 词：骨髓间充质干细胞 分离培养 鉴定 犬 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学]