体外全骨髓贴壁法培养人骨髓间充质干细胞的实验研究  

Experimental Study on Whole Bone Marrow Adherent Culture of Human Bone Marrow Mesenchymal Stem Cells

作　　者：温呈洪[1] 华强[1] 雷鸣鸣[1] 严光建[1] 黄家骏[1] 邓水平[1] 宋维君[1] 

机构地区：[1]成都体育学院附属体育医院骨科,成都610041

出　　处：《西南军医》2015年第4期381-384,共4页Journal of Military Surgeon in Southwest China

摘　　要：目的探讨体外采用全骨髓贴壁法分离培养人骨髓间充质干细胞(human bone mesenchymal stem cells,h BMSCs)的可行性,为研究h BMSCs提供实验细胞来源。方法从因创伤导致骨折需行骨折复位及髂骨植骨术的志愿者中,获取骨髓,采用全骨髓贴壁法培养和分离h BMSCs,在倒置相差显微镜下观察细胞生长形态,取P3代细胞行表型鉴定;取P1、P2、P3代细胞,分别用胰蛋白酶消化,以培养时间为横轴,细胞数量为纵轴,绘制h BMSCs生长曲线图。结果经全骨髓贴壁法培养分离获得了纯度较高的h BMSCs,细胞呈均一的长梭形、短梭形及多角形,漩涡状或辐射状结构,绘制的生长曲线呈S形。P3代h BMSCs经流式细胞鉴定:CD29、CD44、CD90、CD105分别为99.6%、100%、99.4%、92.5%,呈阳性表达;而表达CD34、CD45分别为0.5%、0.6%,呈阴性表达。结论体外应用全骨髓贴壁培养法可成功分离h BMSCs,方法操作简单易行、经济,且能最大限度的保持细胞活力。Objective To explore the feasibility of whole bone marrow adherent culture of human bone marrow mesenchymal stem cells （hBMSCs） in vitro. Methods The bone marrow was collected from the volunteers with traumatic fracture being reduced or from the volunteers receiving ilium graft; whole bone marrow adherent method was applied in culturing and isolating hBMSCs; inverted mi- croscope was used in observing the cell morphology and the cells of the 3rd generation was taken for cell phenotype identification; the cells of the first, the second and the third generation were taken and digested by trypsin respectively and hBMSCs growth curve was drawn with the culture time as the horizontal axis and the cell number as the longitudinal axis. Results hBMSCs cultured and isolated by whole bone marrow adherent method were of higher purity; the cells had long and short fusiform or in polygon, or in whorls or radial structure, the growth curve showed a typical ＂S＂ type; hBMSCs of the third generation were identified by flow cytometry： CD29, CD44, CD90 and CD105 were positive with the percentage of 99.6%, 100%, 99.4% and 92.5% respectively while CD 34 and CD45 were nega- tive with the percentage of 0.5% and 0.6% respectively. Conclusions Whole bone marrow adherent culture of hBMSCs in vitro is of easy operation and lower cost, more over, it can maintain maximum cell viability.

关 键 词：骨髓间充质干细胞 分离培养 全骨髓贴壁法 细胞培养 

分 类 号：R322.2[医药卫生—人体解剖和组织胚胎学]