摘要
目的研究人骨髓间充质干细胞(MSCs)的分离和培养条件,并探讨其生物学特性.方法取无恶性血液病的32～65岁的成人骨髓7例,经密度梯度离心得到单个核细胞,加入含10%胎牛血清的DMEM/F12培养液,将细胞的密度调整到1×106个/mL,接种到12孔培养板中(1 mL/孔)进行培养,48 h 后更换新鲜培养液,以后每3 d 换液1 次.当细胞铺满培养板底90%以上时,按常规方法进行细胞传代培养.用流式细胞术检测培养细胞的CD29、CD44、CD166、CD105、CD34、CD45、HLA-DR表达情况.并且使用脂肪细胞诱导培养液诱导培养MSCs,油红O染色检测其能否被诱导分化为脂肪细胞.结果成人骨髓接种后24 h内细胞开始聚集成细胞集落,并从集落中长出少许长梭形细胞,6～15 d细胞进入快速增殖期,一般培养20～25 d后细胞汇合成片铺满培养板底;而传代培养的细胞每代约需10～15 d即可铺满瓶底.流式细胞术检测结果显示,此次培养的骨髓来源细胞高表达CD29、CD44、CD166、CD105,不表达CD34、CD45、HLA-DR,符合骨髓MSCs细胞的特性.油红O染色显示MSCs可以被诱导分化为脂肪细胞.结论用密度为1.077 g/mL淋巴细胞分离液作为分离液,密度梯度离心法分离培养的人骨髓MSCs增殖力强,操作简单,是一种较好的分离和培养MSCs的方法.MSCs可以被诱导分化为脂肪细胞.
Objective To study a method for the isolation and culture of human bone marrow mesenchymal stem cells （MSCs） in vitro and explore their biological properties. Methods Bone marrow aspiration samples were obtained from 7 adult human donors 32 to 65 years old without severe hematopietic diseases. Mononuclear cells were acquired after density gradient centrifugation of the collected bone marrow aspiration. Then they were seeded at 1× 10^6 cells/ml in DMEM/F12 supplemented with 10% fetal bovine serum （FBS） in a hole of 12 holes culture plate. The medium was changed first time after 48 hours, and once every 3 days thereafter. When the cultures reached nearly 90% of confluence, cells were passaged with routine methods. Cell surface antigens which contain CD29, CD44, CD166, CD105, CD34, CD45, HLA-DR were detected with flow cytometry. MSCs were treated with adipocyte culture fluid, then they were detected with oil red stain to ascertain whether MSCs could be induced to adipocyte or not. Results A small percentage of isolated cells were adherent to the flasks and grew as typically fibroblas-tic shape 48 hours after plating. Primary cells derived from adult human bone marrow reached 90% of confluence in20 to 25 days. Passaged cells reached 90% of confluence in 10 to 15 days. The cultured adherent cells typically expressed CD29, CD44, CD166 and CD105, while CD34, CD45; HLA-DR of them were negative. The result of oil red stain showed that MSCs were able to be induced to adipocyte. Conclusion The isolated and cultured MSCs by the method of density gradient centrifugation are of Singular configuration , stable growth and rapid proliferation. This method is a good one to isolate and culture MSCs. MSCs can be induced to adipocyte.
出处
《江西医学院学报》
2005年第6期1-4,F0004,共5页
Acta Academiae Medicinae Jiangxi
基金
江西省科委重大科技攻关资助项目(200301003)
关键词
骨髓
间充质干细胞
细胞培养
bone marrow
mesenchymal stem cells
cell culture
