骨髓源性神经样细胞体外存活、增殖的特性  

Proliferation and survival of mesenchymal stem cell-derived neuron-like cells in vitro

作　　者：李涛[1] 洪光祥[1] 李进[1] 陈振兵[1] 康皓[1] 王发斌[1] 冯亚高[1] 黄启顺[1] 翁雨雄[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院手外科,湖北省武汉市430022

出　　处：《中国组织工程研究与临床康复》2007年第15期2846-2849,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:寻找骨髓间充质干细胞体外诱导分化为神经元样/星形胶质样细胞的有效方法,并评价其体外存活和增殖特性。方法:实验于2005-07/2006-08在华中科技大学同济医学院附属协和医院完成。采用贴壁筛选法分离培养大鼠骨髓间充质干细胞。收集第5代骨髓间充质干细胞单细胞悬液,以1×109L-1置入6孔培养板中,加入DMEM+20%胎牛血清生长培养基,24h后更换生长培养基为DMEM/F12+2%B27+40μg/L碱性成纤维生长因子+20μg/L内皮生长因子诱导分化培养基,诱导10～20d,即采用多因子分阶段逐步诱导方法定向诱导骨髓间充质干细胞分化为神经前体样细胞。更换诱导分化培养基为DMEM/5%胎牛血清生长培养基,分两组进行诱导:一组向神经元样诱导分化:全反式维甲酸连续加样,首次浓度0.5μmol/L,以后每天全量加样;二组向星形胶质样细胞诱导分化:10μg/L血小板衍生生长因子BB首次加样,以后每天半量加样,均连续诱导10～14d,进而分化为神经元样/星形胶质样细胞。应用活细胞计数试剂盒CCK-8检测神经元样/星形胶质样细胞的增殖及存活情况,绘制生长曲线及细胞存活曲线。结果:①碱性成纤维生长因子+表皮生长因子连续诱导7d后骨髓间充质干细胞分化为球团样的神经前体样细胞,巢蛋白表达阳性。②全反式维甲酸、血小板衍生生长因子BB分别连续诱导10d后骨髓间充质干细胞逐步分化出神经元样、星形胶质样细胞,并分别表达纤维丝蛋白200及胶质纤维酸性阳性蛋白。③神经元样细胞早期生长较缓慢,具有一定的增殖能力,六七天后达到高峰,此后增殖能力明显下降,细胞存活率明显下降,十四五天之后细胞存活率下降到7.6%以下,难以传代培养。星形胶质样细胞初期生长慢,5d后生长加速,进入对数生长期,七八天后进入平台期,仍保持较高的细胞存活率。结论:采用多因子分阶段诱导方法可成功诱导骨AIM： To search for the effective method of mesenchymal stem cells （MSCs） differentiating into neuron-like cells and astrocyte-like cells in vitro and evaluate the proliferation and survival capacity of MSCs-differentiated cells. METHODS： The experiment was performed at the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from July 2005 to August 2006, By adherence Screening method, MSCs of rats were isolated and cultured, and then monoplast suspension of the 5^th passage MSCs was collected, put in 6-well culture plate at 1×10^9 L^-1 and DMEM plus 20% fetal calf serum （FCS） medium was added. 24 hours later, growth medium was changed into DMEM/F12＋2%B27＋40 μg/L basic fibroblast growth factor （bFGF）＋20 μg/L epidermal growth factor （EGF）, inducing for 10-20 days, namely, MSCs were differentiated into nerve precursor-like cells by multi-cytokines induction protocols. Medium was changed into DMEM/5% FCS growth medium, and MSCs were induced in two groups： In group one MSCs were differentiated into neuron-like cells, all-trans retinoic acid at 0.5 μmol/L firstly, and then sampling of full dose every day; In group two MSCs were differentiated into astrocyte-like cells： 10 μg/L platelet-derived growth factor （PDGF） BB was added firstly, and then added a half dose, induced for 10-14 days successively. Cell counting Kit CCK-8 was applied to measure the proliferation and survival of neuron-like cells/astrocyte-like cells, and growth curve and survival curve were drawn. RESULTS： ①In the presence of selected cytokines MSCs were effectively differentiated into neurospheres-like cells with EGF and bFGF for 7 days. Nidogen expressed positive.②Then neurospheres-like cells were differentiated into neuron-like cells with retinoic acid and astrocyte-like cells with PDGF-BB for over 10 days, positive for nestin, NF200 and glial fibnllary acidic protein （GFAP） immunohistochemistry staining. ③Neuron-like cells grew slowly in eady phase, with a cer

关 键 词：间充质干细胞 细胞因子 诱导分化 神经元样细胞 星形胶质样细胞 

分 类 号：R394.2[医药卫生—医学遗传学]