大鼠骨髓间充质干细胞体外分离、纯化与培养适宜条件的筛选  

Suitable conditions for the isolation,purification and culture of rat bone marrow mesenchymal stem cells in vitro

作　　者：庄淑波[1] 刘毅[1] 陈克明[1] 肖斌[1] 

机构地区：[1]解放军兰州军区兰州总医院烧伤整形外科,甘肃省兰州市730050

出　　处：《中国组织工程研究与临床康复》2007年第20期3886-3891,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：全军"十一五"医学科研面上项目(06MA079);甘肃省自然科学基金(3zs061-A25-099)资助项目~~

摘　　要：目的:为获得大量、高纯度的骨髓间充质干细胞,筛选体外分离、纯化和培养的适宜条件。方法:实验于2006-08/2007-07在解放军兰州军区兰州总医院进行。①实验材料:2月龄SD大鼠,雄性,体质量(100±20)g,由解放军兰州军区兰州总医院实验动物科提供。②实验方法:分别采用全骨髓法(贴壁筛选法)和密度梯度离心法分离和纯化大鼠骨髓间充质干细胞。将采用密度梯度离心法获得的单个核细胞按1×107,1×108,1.5×108,2×108,1×109,2×109L-1的接种密度分别分成1～6组,接种在24孔板内,每组接种6孔。比较细胞出现伸展时间、原代培养时间。于细胞接种后第1,2,3,4,5,6天进行首次换液。将换下的部分细胞置于新的培养皿中进行培养,至少培养3d,观察是否还有新的贴壁细胞出现,以确定首次换液时间。观察体积分数为0.10,0.12,0.15,0.18,0.20血清对原代及传代后细胞生长的影响,比较不同浓度血清中细胞数量、集落形成率。集落形成率=集落数目/接种细胞数目×100%。采用流式细胞仪检测骨髓间充质干细胞细胞周期。选取第5代扩增的骨髓间充质干细胞,接近完全融合后进行成骨和成脂肪诱导培养。诱导剂分别为:10nmol/L地塞米松、0.05nmol/L抗坏血酸及10nmol/Lβ-甘油磷酸钠;1μmol/L地塞米松、0.5mmol/LIBMX、0.01mmol/L人胰岛素、0.01mmol/L吲哚美辛。并设未加诱导剂培养液培养的骨髓间充质干细胞对照。③实验评估:采用碱粒酶测定成骨诱导后细胞,采用油红O染色鉴定成脂肪诱导后细胞。结果:①细胞出现伸展时间、原代培养时间:密度梯度离心法培养细胞出现伸展时间与全骨髓法相比,差异无显著性意义。密度梯度离心法原代培养时间较全骨髓法长[(9.41±1.11),(14.73±2.86)d,P<0.05]。②不同接种密度对密度梯度离心法所获细胞生长影响:1×109L-1组细胞出现伸展时间及原代培养细胞融合时间较早。1×107L-1组培AIM： To explore the suitable conditions for isolation, purification and culture of rat bone marrow mesenchymal stem coils （MSCs） in vitro. METHODS： The experiment was conducted in the General Hospital of Lanzhou Military Area Command of Chinese PLA between August 2006 and July 2007. Two-months-old male SD rats of （100±20） g were provided by the Experimental Animal Section of Lanzhou Military Area Command of Chinese PLA, and with the whole bone marrow, namely adhesive screening method and density gradient contrifugation method, MSCs were isolated and purified, Then the cells isolated by the latter method were seeded onto the 24-weell plates at concentrations of 1 ×10^7, 1×10^8, 1.5×10^8, 2×10^8, 1 ×10^8, and 2×10^9 L^-1 with 6 wells for each group, Extension time and the primary generation culture time were compared. The culture fluid was firstly changed after the cells were incubated for 1, 2, 3, 4, 5, and 6 days, respectively, The partial cells exchanged were cultured in another culture dish for at least 3 days to observe whether the cells attached or not to determine the first time for the fluid exchange. The effect of volume fraction 0.10, 0.12, 0,15, 0.18, and 0.20 serum on the proliferation of primary and cultured cells to compare the cell number and colony forming efficiency in different density serums： colony forming effficiency=number of colony/coil number of incubation×100%. The cell cycle of MSCs was detected by flow cytometry. The fifth generation MSCs were selected and induced to bone formation and fat formation after completely fusion by 10 nmol/L dexamethasone （DXM）, 0.05 nmol/L antiscorbic acid, and 10 nmol/L 13 sodium glycerophosphate; and 1 μmol/L DXM, 0.5 mmol /L IBMX, 0.01 mmol/L human insulin, and 0.01 mmol/L indomethacin. Meanwhile, the MSCs cultured in medium without inductor served as control. The cells after induction were detected by diazo coupling reaction, and cells after fat induction were identified by oil red O dyeing. RESULTS： （1）The extension time o

关 键 词：骨髓间充质干细胞  细胞培养  大鼠 

分 类 号：R394.2[医药卫生—医学遗传学]