人骨髓间充质干细胞体外分离培养及其生物学特性  

The Isolation and Characterization of Human Bone Marrow Mesenchymal Stem Cells

作　　者：郑有华[1] 何滔[2] 匡世军[1] 张志光[1] 苏凯[1] 

机构地区：[1]中山大学光华口腔医学院口腔颌面外科,广州510055 [2]中山大学口腔医学研究所,广州511080

出　　处：《国际医药卫生导报》2010年第2期129-134,共6页International Medicine and Health Guidance News

基　　金：广东省科技厅社会发展项目(2008B030301312)

摘　　要：目的探讨人骨髓来源MSCs的分离、培养和生物学特性,为骨组织工程提供种子细胞。方法从捐赠者髂骨穿刺收取骨髓细胞,采用密度梯度离心和贴壁培养法进行培养和纯化BMSCs,取生长良好的P3或P4代BMSCs进行检测:①MTT法测定细胞增殖和生长曲线分析;②流式细胞仪检测BMSCs细胞表面抗原标记和细胞增殖周期;③免疫荧光分析细胞骨架;④细胞染色体核型分析;⑤成骨细胞诱导分化及检测。结果①利用密度梯度离心和贴壁筛选的方法分离人BMSCs,经传代后细胞形态呈梭形,均匀一致。②MTT法测定细胞增殖和生长曲线分析结果显示BMSCs在传代后经历潜伏期、对数生长期和平台期。③流式细胞仪检测显示所获得的BMSCs表面抗原标记高度一致。④BMSCs细胞周期检测结果为G0+G1期91.3%、G2期4.1%和S期4.6%。⑤微管微丝免疫荧光染色结果显示,培养的BMSCs具有良好的细胞骨架系统。⑥第7代的BMSCs染色体检测结果显示,被测标本有正常染色体数目(2n=46,XY),且未见染色体异常。⑦成骨诱导后,Von kossa和茜素红染色均呈阳性。结论从人骨髓中能分离出高度一致的BMSCs,这些细胞具有正常细胞骨架结构以及正常的人类染色体数目和形态,具有向成骨细胞分化能力,可作为骨组织工程的种子细胞。Objective To investigate the methods of the isolation,cultivation and characteristic of mesenchymal stem cells derived from human bone marrow（hBMSCs）,so as to pave the way for searching the new source of seed cells in bone tissue engineering.Methods Bone marrow were inspired from ilium of donators,mononuclear cells were isolated by density gradient centrifugation and cultured in vitro.The higher purity of BMSCs were obtained by keeping the adherent cells and removal of nonadherent cells repeatedly.P3 or P4 BMSC populations were collected and examined as follow：①drawing a growth curve of the BMSCs by MTT colorimetric assay;②detecting expressed the surface marker expression and the growing circle of BMSCs by flow cytometric analysis;③Cytoskeleton was detected by immunofluorescence;④Karyotype of BMSCs was examined by Giemsa stain.⑤the efficiency of differentiation into osteoblast was assessed byrelated methods.Results The highly purified BMSCs were obtained by density gradient centrifugation andkeeping the adherent cells derived from human bone marrow and the BMSCs showed long fusiform or spindleand fibroblast-like shaped after repeated passages.？The growth curve by MTT displayed that every passageexperienced incubation period,log phase and platform period.？The cell surface markers assay with flowcytometry indicated highly uniform of surface markers.？The cell circle assay displayed G0 ＋ G1 stage was inhigh percentage（about 91.3%）,but G2 and S stage were in low percentage（about 4.1% and 4.6%respectively）.？ The cultured BMSCs possessed normal morphology and cytoskeleton.？The cells of P7 hadnormal human chromosome number（46,XY） and appearance.⑦Under inductive medium in vitro,the BMSCs could be induced into osteoblasts which were positive when stained by Von kossa and alizarin red.Conclusions： The highly purity and uniform of MSCs derived from human bone marrow can be obtained These BMSCs possess normal morphology,cytoskeleton,and chromosome number,the ability of differentiati

关 键 词：骨髓 间充质干细胞 细胞分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]