婴幼儿骨髓间充质干细胞体外培养及基本生物学特性的实验研究  

An Experimental Study on Cell Culture in vitro and Basic Biological Character of Infant Bone Marrow Mesenchymal Stem Cells

作　　者：邢万红[1] 徐志伟[2] 陆兆辉[2] 董新寨[1] 席虎玉[1] 

机构地区：[1]山西医科大学第二医院,太原030001 [2]上海交通大学医学院附属新华医院上海儿童医学中心,上海200127

出　　处：《中国生物医学工程学报》2007年第1期144-148,共5页Chinese Journal of Biomedical Engineering

摘　　要：目的建立一种体外分离和培养婴幼儿骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的方法,并探讨其基本生物学特性。方法麻醉、无菌条件下行胸骨穿刺抽取先天性心脏病婴幼儿骨髓3mL^5mL,经密度梯度离心获得单个核细胞(Percoll分离液,γ为1.073),接种入含10%胎牛血清的低糖DMEM培养液,测定其生长曲线,流式细胞仪检测婴幼儿BMSCs表面标记,并观察其形态、贴壁率、集落形成能力和超微结构的变化。结果培养的婴幼儿BMSCs 3h^4h后开始贴壁,贴壁率为65%~80%;2d^3d可见集落形成;10d左右汇合90%以上。BMSCs随着传代次数增加集落形成率逐渐降低。培养期间细胞形态均一,基本上为梭形成纤维细胞样形态。婴幼儿BMSCs表面标记CD29、CD44、CD71、CD90表达阳性,CD3、CD14、CD34、CD45、HLA-DR不表达。透射电镜观察,婴幼儿BMSCs具有BMSCs典型的超微结构。结论成功地建立了一种体外分离培养先天性心脏病婴幼儿BMSCs的方法,获得的细胞形态均一,生长稳定,增殖较快,能为体外构建组织工程化先天性心脏病修复材料提供重要的种子细胞的来源。Objective To establish a method for the isolation and culture of infant bone marrow mesenchymal stem cells （BMSCs） in vitro, and study some of their basic biological properties. Methods Three to five milliliters bone marrow were extracted from the sternum of infant with congenital heart disease when anesthesia and sterile conditions. Mononuclear cells were obtained by density gradient centrifugation（ Percoll, γ 1.073）, then inoculated in the low glucose DMEM culture medium containing 10% fetus bovine serum. Growth curve was made, the surface markers of infant BMSCs were detected by flow cytometer. The Changes of their shapes, anchoring rate, colony formation rate and uhrastructure were observed. Results Infant BMSCs began to anchor after cultured for 3h - 4h, anchoring rate is 65% -80%. Formation of colony could be observed on the 2^nd to 3^rd day. More than 90% infant BMSCs were confluent at 10^th day. The cultured cells could be passaged continuously, and their colony formation were gradually decreased with passage number increased. The cells＇ shape was uniform during their culture period, basically fusiform shape or fibroblast-sbaped. The surface markers of infant BMSCs analysis indicated the positive expression of CD29, CD44,CD71 ,CD90,and no expression of CD3 ,CD14,CD34,CD45 ,HLA-DR. By observing with transmission electron microscopy, the infant BMSCs showed the typical ultrastructure of BMSCs. Conclusions We have established successfully a method for the isolation and culture of infant BMSCs in vitro. The shape of obtained cells is uniform,their growth is stable and proliferation is rapid, It can provid the important resource of seed cells in constructing tissue engineered material in vitro for congenital heart disease.

关 键 词：婴幼儿 骨髓间充质干细胞 细胞培养 增殖 

分 类 号：R318[医药卫生—生物医学工程]