C57/BL6小鼠骨髓间充质干细胞分离培养方法的改良和鉴定  

The biological characteristics of C57/BL6 mice bone marrow-derived mesenchymal stem cells isolated and cultured with attachment method

作　　者：夏宇[1] 盛瑾[2] 朱铠[3] 过常发[3] 王春生[3] 

机构地区：[1]遵义医学院附属医院胸心血管外科,563003 [2]遵义医学院附属医院心血管内科,563003 [3]上海复旦大学中山医院心血管外科,200032

出　　处：《贵州医药》2012年第9期779-782,共4页Guizhou Medical Journal

摘　　要：目的建立一种体外纯化及培养扩增C57/BL6小鼠骨髓间充质干细胞(MSCs)的简单有效方法。方法利用骨髓MSCs在体外培养皿中培养时具有贴壁生长的特性,采用全骨髓法分离培养C57/BL6小鼠MSCs,通过体外培养和连续传代达到纯化和扩增MSCs目的,并与以优化。倒置显微镜下观察细胞形态变化情况;利用四唑盐比色(MTT)法测定MSCs的生长曲线;流式细胞仪(FCM)鉴定MSCs膜抗原。结果 C57/BL6小鼠胫骨全骨髓细胞在分离后与含15%优质胎牛血清DMEM/F12培养基混合,约5～10min后即可见大量有核细胞贴壁,细胞形态为圆形,24～48h后可见贴壁细胞转变为椭圆形、多角形及短梭形,7～12d时细胞呈长梭形并达到90%单层融合。通过传代细胞进一步纯化及扩增。细胞传代后2d内处于潜伏期,3d后进入生长期,5d后进入平台期。第4代MSCs进行FCM检测CD45、CD44、CD29和CD54阳性率分别为2.4%、83.2%、95.1%和94.1%。激光免疫共聚焦结果显示造血细胞表面标物CD34和CD45阴性,而CD44和CD105阳性。结论 C57/BL6小鼠全骨髓贴壁法能有效得到分离纯化的MSCs,用此方法培养的C57/BL6小鼠MSCs生长稳定,增殖能力活跃,具有MSCs的一般生物学特性,可以为组织工程提供理想的种子细胞。Objective To establish a simple and effective method of isolation,purification and cultivation of C57/BL6 mice bone marrow-derived mesenchymal stem cells（MSCs） in vitro,and explore their biological characteristics. Methods MSCs were isolated and purified for culture in vitro and serial passage by the attachment culture method.The morphological changes of the MSCs were continuously observed under the inverted microscope.The cell growth curve was measured by MTT method,and membrane antigens were detected with flow cytometry（FCM）. Results The MSCs in primary culture which were adhered to plastic surface within 48 hours after displacement took the oval,asteroid,short and fusiform shapes.7 to 12 days after culture in DMEM/F12 medium containing 15% FBS,the cells reached 90% confluence in single layers,taking a long and fusiform shape.Further purification was achieved by expansion at serial passages.MSCs were in latency for 2 days,converted into growth period on the 3rd day and entered the stationary phase on the 5th day.FCM results indicated that the positive rate of CD45,CD44,CD29 and CD54 was 2.4%,83.2%,95.1% and 94.1%,respectively. Conclusion The attachment culture method can be effectively used to isolate and purify C57/BL6 mice MSCs.The cultured MSCs are stable in growth with active proliferation and share the general biological characteristics of MSCs,which will further make them ideal seed cells for tissue engineering.

关 键 词：间充质干细胞 骨 髓 贴壁法分离培养 

分 类 号：R446.8[医药卫生—诊断学]