首次换液时间对贴壁法培养骨髓间充质干细胞纯度及增殖的影响  

Influence of the timing of the first medium exchange on the purity and proliferation of bone mesenchymal stem cells cultured by attachment method

作　　者：杨辉[1] 蔡光先[1] 刘柏炎[1] 蔡莹[1] 谢勇[1] 

机构地区：[1]湖南中医药大学内科实验室

出　　处：《中国组织工程研究与临床康复》2007年第20期3868-3871,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：湖南省自然科学基金资助(03JJY3040)~~

摘　　要：目的:观察不同时间首次换液对贴壁法培养骨髓间充质干细胞形态和表面标志物表达的影响。方法:实验于2005-09/2006-12在湖南中医药大学内科实验室完成。实验材料:三四周龄昆明种小鼠由湖南中医药大学实验动物室提供(许可证号:医动字第20-002号)。实验方法:无菌条件下分离小鼠股骨,以低糖DMEM培养液冲出骨髓,1000r/min离心10min,去除脂肪和上清液,DMEM重悬,洗涤细胞,同样条件下离心,弃上清液,将获得的骨髓间充质干细胞用含体积分数为0.15胎牛血清、105U/L青霉素G、100mg/L链霉素的低糖DMEM完全培养基重悬沉淀,以1×108L-1接种于24孔板,置于37℃、体积分数为0.05的CO2饱和湿度培养箱中培养。第1组于24h全量换液,第2组于3d全量换液,第3组于5d全量换液。比较骨髓间充质干细胞在原代培养24h、3d、5d首次换液后细胞形态、生长曲线和表面标志物表达。结果:①骨髓间充质干细胞形态:原代培养24h换液,可见少量贴壁细胞,形态不规则。原代培养3d首次换液,可见较多贴壁细胞,并形成细胞团,细胞为椭圆形、短梭形。原代培养5d换液,可见明显集落形成,贴壁细胞量多,细胞呈梭形。②骨髓间充质干细胞生长曲线:原代培养24h首次换液,骨髓间充质干细胞生长较缓慢,对数生长期晚;原代培养3d首次换液,骨髓间充质干细胞增殖速度快,对数生长期早;原代培养5d首次换液,骨髓间充质干细胞前6d生长速度较快,6d后生长渐缓慢。③骨髓间充质干细胞表面抗原:三组间比较CD44、CD45阳性表达率差异均具有显著性意义[24h首次换液:(88.13±1.60)%,(26.25±2.60)%;3d首次换液:(85.21±1.80)%,(29.37±1.30)%;5d首次换液:(79.85±1.50)%,(36.54±1.20)%,P<0.01]。结论:不同时间首次换液对贴壁法骨髓间充质干细胞的生长有影响,单从细胞纯度来考虑,24h首次换液较为理想,从细胞生长情况和纯度综合考虑,3d首次换液较为理想。AIM： To study the influence of the first exchange of medium at different time on the cellular morphologic and surface marker of bone mesenchymal stem cells （MSCs） cultured by attachment method. METHODS： The experiment was carried out in the laboratory of Department of Internal Medicine, Hunan University of Traditional Chinese Medicine from September 2005 to December 2006. Three to four weeks old Kunming mice were provided by the experimental animal center of Hunan University of Traditional Chinese Medicine （No. 20-002）. Under sterile conditions, the femur bones were isolated from the mice, and the bone marrow was washed out with low glucose DMEM, 1 000 r/minute and centrifuged for 10 minutes to remove the fat and supernatant. The remainder was suspended and washed again, to remove the supernatant, and the obtained MSCs were resuspended in LG-DMED complete culture medium supplemented with volume fraction 0.15 fetal bovine serum, 105 U/L penicillin G, and 100 mg/L streptomycin and seeded onto a 24-well plate at the concentration of 1×10^8 L^-1; then the cells were cultured in a humidified incubator with volume fraction 0.05 CO2 at 37 %. The medium of the first group was all changed after 24 hours; the second group was all changed after 3 days; the third group after 5 days. The cell morphous, growth curve and surface marker expression were compared with medium changed at different time.RESULTS： （1）MSCs morphous： In the first group that the medium was firstly exchanged after primary culture for 24 hours, few cells adhering to culture plastic were observed with irregular cell shape. When the medium was firstly exchange after 3 days, many cells adhering to culture plastic ware found, and formed cell group; the cells were oval-shaped and short spindle-shaped. In the third group, conspicuous colonies were observed and the cells were spindle-shaped. （2）MSCs growth curve： The cells cultured in medium first exchanged in 24 hours slowly grew, with late logarithmic phase. The cells in the second gro

关 键 词：骨髓间充质干细胞 细胞培养技术 表面标志 小鼠 

分 类 号：R394.2[医药卫生—医学遗传学]