成人骨髓间充质干细胞体外低氧培养及生物学特征  

IN VITRO HYPOXIC CULTURE OF HUMAN MARROW MESENCHYMAL STEM CELLS AND THEIR BIOLOGICAL FEATURES IN ADULTS

作　　者：王庆德[1] 杨述华[1] 杨操[1] 叶哲伟[1] 胡勇[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中国修复重建外科杂志》2007年第10期1128-1132,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30170945)~~

摘　　要：目的建立人骨髓间充质干细胞(human marrow mesenchymal stem cells,hMSCs)体外低氧培养及向成骨细胞分化的生物模型,研究其生物学特征,为骨组织工程提供实验依据。方法采用人淋巴细胞分离液分离健康成人hMSCs,DMEM-LG(含20%胎牛血清)中培养,10～14d后传代培养。取第2代细胞,根据培养氧浓度及培养基类型分为4组:正常氧组(20%O2加DMEM-LG,A组)、低氧组(1%O2加DMEM-LG,B组)、正常氧成骨诱导组(20%O2加条件培养基,C组)及低氧成骨诱导组(1%O2加条件培养基,D组),通过细胞计数、细胞增殖测定(MTT法)、集落形成检测、RT-PCR检测、碱性磷酸酶(alkaline phosphatase,ALP)活性检测及茜素红染色,研究低氧环境对hMSCs生物学行为的影响。结果与A组相比,B组及D组中的hMSCs有较高的增殖速度,并且不受条件培养基的影响,比较差异有统计学意义(P<0.01)。B组中的hMSCs生成的集落逐渐增多,A组9d后生成的集落数基本不发生变化。D组培养的hMSCs的ALP活性逐渐增高,但明显低于C组,两者差异有统计学意义(P<0.01)。定量RT-PCR检测,C组ALP、骨钙素、Ⅰ型胶原蛋白及骨粘连蛋白mRNA表达量明显增加(P<0.01)。培养4周后,与其他3组相比,C组可见到明显的钙盐沉积和染成红色的钙结节。结论低氧使hMSCs增殖率增加,集落形成能力增强,抑制向成骨细胞分化。Objective To establish a model of the human marrow mesenchymal stem cells （hMSCs） cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia. Methods The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects, hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20% fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types： the normoxic group （20%02, DMEM-LG, Group A）, the hypoxic group（1%O2, DMEM-LG,Group B）, the normoxic osteoblast induction group （20%O2, conditioned medium, Group C）, and the hypoxic osteoblast induction group （1%O2, conditioned medium, Group D）. The biological features of the cultured hMSCs under hypoxia were assessed by the cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase （ALP） activity, and the alizarin red staining. Results The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A （P〈0.01）, and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A（P〈0. 01）. After the induction, hMSCs in the Group B had a decreased number of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increased activity of ALP, which was significantly lower than that in the Group C（P〈0.01）. The RT-PCR examination revealed that ALP, osteocalcin, and mRNA expressions of collagen type Ⅰ and osteonectin in the Group C significantly increased （P〈0. 01）. By comparison among the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained

关 键 词：骨髓间充质干细胞 缺氧 成骨细胞 细胞培养 

分 类 号：Q813.11[生物学—生物工程] R318[医药卫生—生物医学工程]