小鼠骨髓间充质干细胞分离培养新方法  

A New Method for Isolating and Culturing Mouse Bone Marrow Mesenchymal Stem Cells

作　　者：杨燕美[1] 李红[2] 张雷[2] 党瑞杰[1] 李萍[1] 王晓燕[2] 朱恒[2] 郭希民[2] 张毅[2] 刘元林[2] 毛宁[2] 江小霞[2] 温宁[1] 

机构地区：[1]中国人民解放军总医院口腔科,北京100853 [2]中国人民解放军军事医学科学院基础医学研究所,北京100850

出　　处：《中国实验血液学杂志》2013年第6期1563-1567,共5页Journal of Experimental Hematology

基　　金：国家重点基础研究发展计划"973计划"(编号2010CB529904);"863"重大专项(编号2011AA020114);国家自然科学基金面上项目(编号81271936;81070393;81170523;81170461;31170926)

摘　　要：本研究旨在建立一种方便、快捷、有效体外分离培养小鼠骨髓间充质干细胞(mesenchymal stem cells,MSC)的方法。在无菌条件下取小鼠股骨、胫骨,分别用全骨髓贴壁法、骨片消化法、骨髓加骨片法分离小鼠MSC,比较MSC集落出现时间、大小及数量;应用流式细胞术检测细胞免疫表型,并进行MSC成骨、成脂分化鉴定。结果表明,骨髓加骨片法出现的集落时间最早,集落数量最多,第4天集落数为20±4个;骨片消化法为11.5±2.5个;全骨髓贴壁法最少,为9.5±1.5个;到第3代时,骨髓加骨片法获得细胞数量最多,是全骨髓贴壁法和骨片消化法的2倍;流式细胞术检测结果显示,获得的细胞高表达干细胞标志之一Sca-1,高表达CD44、CD29,不表达白细胞表面抗原CD45和内皮细胞标志CD31等;所分离的MSC在成骨诱导体系和成脂诱导体系中能分别向成骨细胞和脂肪细胞分化,具有典型的MSC特性。结论:骨髓加骨片法是一种方便、快捷、有效的小鼠骨髓MSC分离方法。This study was purposed to establish a convenient and efficient method for isolating and culturing mouse bone marrow mesenchymal stem cells （MSC）. The femurs and tibias of mouse were taken under sterile condition. MSC were isolated and cultured with flushing- out bone marrow or collagenase-digested bone fragment or bone marrow plus bone fragment. MSC colony number and size were compared. Immunophenotype and differentiation ability were tested to identify MSC. The results showed that colonies from bone marrow plus bone fragment group came out earliest and the colony number was 20 ± 4 at day 4 ; there were 11.5 ±2.5 colonies in collagenase-digested bone fragment group and 9. 5± 1.5 in flushing- out bone marrow group. The total cell yields of MSC after passaging showed best in bone marrow plus bone fragment group. Flow cytometry data showed the cultured cells expressed Sca-1, CD44 and CD29, not expressed pan-leukocyte surface marker CD45 and endothelial cell marker CD31. The isolated and cultured MSC could differentiate into osteoblast at the osteogenic differentiation condition, or adipocyte at adipogenic differentiation condition. It is concluded that the method of bone marrow plus bone fragment is convenient and efficient for isolating and culturing MSC.

关 键 词：间充质干细胞 骨髓加骨片法 小鼠 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]