人骨髓间充质干细胞分离方法的比较

Comparison of separation methods of human mesenchymal stem cells

目的:探讨人骨髓间充质干细胞原代培养过程中最佳的分离方法。方法:留取暨南大学第一附属医院骨科2004-10/2005-06健康供者的骨髓10份,分别采用羟乙基淀粉沉淀法,淋巴细胞分层液分离法,氯化铵裂解红细胞法,全骨髓法四种方法分离骨髓有核细胞,观察四种方法在细胞出现伸展的时间,原代培养的时间方面的差异,流式细胞仪对培养得到的间充质干细胞进行表面标志检测。结果:①在其他条件相同的情况下,羟乙基淀粉沉淀法10份标本全部培养成功,淋巴细胞分层液分离法10份中有9份得到间充质干细胞,氯化铵裂解红细胞法10份中仅1份得到间充质干细胞,全骨髓培养法10份中有3份得到间充质干细胞。②羟乙基淀粉沉淀法细胞出现伸展和原代培养的时间短于淋巴细胞分层液分离法犤乙基淀粉沉淀法:羟(54.0±3.0h,(12.4±1.4)d;淋巴细胞分层液分离法:(74.0±5.2)h,(14.6±0.9)d,)P<0.01犦③流式细胞仪检测骨髓间充质干细胞表面表达CD13,CD29,。CD44,不表达造血细胞的标志CD45,CD34。结论:经比较羟乙基淀粉沉淀法明显优于其他分离方法,在人骨髓间充质干细胞的培养过程中建议使用此种物理沉降方法分离骨髓有核细胞。

AIM： To investigate the proper methods of separating mesenchymal stem cells （MSCs） from bone marrow in primary culture. METHODS＂ Ten bone marrow samples of healthy donors were gained from Department of Orthopaedics, First Affiliated Hospital, Jinan University between October 2004 and June 2005. Bone marrow karyotes were separated with four methods： hespan method, Ficoll-Hypaque method, NH4Cl solution method and complete bone marrow blood method, respectively. The time of stretching and time of primary culture were compared with the four methods. The surface markers were identified by flow cytometry. RESULTS： ①Under the same conditions, ten samples were all cultured successfully by Hespan, nine of 10 by Ficoll-Hypaque, only one of 10 by NH4Cl solution, and three of 10 by complete bone marrow, ②Time of primary culture and stretching time with Hespan were shorter than that with Ficoll-Hypaque [Hespan ： （ 54.0±3.0） hours, （12.4±1,4） days; Ficoll-Hypaque：（74.0±5.2） hours, （14.6±0.9） days, P 〈 0.01]. ③MSCs strongly expressed adhesion molecules CD13, CD29 and CD44, but not antigens of hematopoietic CD34, CD45. CONCLUSION： Hespan is superior to other methods significantly in separating of MSCs by comparison. This physical sedimentation method is a very good method that can be used during the culture of MSCs to separate nucleated cells in bone marrow.