大鼠骨髓间充质干细胞向神经元样细胞诱导分化及神经蛋白的动态表达

Differentiation from rat bone marrow mesenchymal stem cells into neuron-like cells and dynamic expression of neuroprotein

背景:现阶段骨髓间充质干细胞定向诱导分化为神经元样细胞的方法并不统一。目的:探讨大鼠骨髓间充质干细胞体外向神经样细胞诱导分化的条件,及其定向分化过程中神经蛋白的动态表达。设计、时间及地点:细胞形态学观察及蛋白分子水平检测,于2006-09/2007-01在苏州大学生命科学学院完成。材料:清洁级SD大鼠2只。胎牛血清为杭州四季青产品,成纤维生长因子、丁羟基茴香醚为Sigma公司产品,二甲基亚砜为Amresco产品。方法:Percoll法体外分离培养、扩增纯化大鼠骨髓间充质干细胞,调整细胞密度为5×107L-1。设立2组,诱导组用含10%胎牛血清和10μg/L成纤维生长因子的L-DMEM培养基预诱导24h后,再以含2%二甲基亚砜和200μmol/L丁羟基茴香醚的L-DMEM无血清培养基诱导1,3,5h。对照组始终用含10%胎牛血清的L-DMEM进行培养。主要观察指标:细胞形态学变化,免疫荧光染色鉴定神经细胞表型。结果:预诱导24h,胞体由原来的长梭形变成多角形或不规则形,伸出多个短棒状突起,可见2～3个核仁,细胞间漩涡状生长趋势消失,排列无规律;诱导1～3h,细胞逐渐变圆,胞质收缩明显,折光性增强,双级或多极的突起互相连接呈网状;诱导5h,突起出现一、二级分支。诱导1h后部分细胞表达神经前体细胞表面抗原巢蛋白;3h后巢蛋白达高峰,同时表达成熟神经元标志物神经元特异性烯醇化酶和微管蛋白;5h后巢蛋白表达下降,神经元特异性烯醇化酶和微管蛋白达高峰。对照组细胞形态无明显变化,巢蛋白表达为阴性。结论:在添加化学诱导剂之前先使用成纤维生长因子进行预诱导,能促进骨髓间充质干细胞向神经前体细胞和神经元转化,且在诱导分化过程中神经蛋白分子的表达顺序与神经细胞发育过程一致。

BACKGROUND： The method of bone marrow mesenchymal stem cells （BMSCs） differentiation into neuron-like cells are not unified. OBJECTIVE： To investigate the conditions for the neuronal induction of BMSCs from adult rats into neuron-like cells, and the neural markers＇ expression during differentiation. DESIGN, TIME AND SETTING： The cell morphology observation and protein molecular level experiment was performed at the College of Life Science, Soochow University from September 2006 to January ：2007. MATERIALS： Two clean Sprague Dawley rats were used for this study. Fetal bovine serum （Sijiqing, China）, basic fibrohlast growth factor and butyl hydroxy anisol （Sigma, USA）, and dimethyl sulphoxide （Amresco） were used in this study. METHODS： BMSCs were isolated from bone marrow of young adult rats by Percoll method and purified by passage culture. Cell density was regulated to 5× 10^7 L^-1. Cells in the induction group were treated with L-DMEM consisting of 10% fetal calf serum and 10 μg/L basic fibroblast growth factor for 24 hours, then induced with L-DMEM consisting of 2% dimethyl sulphoxide and 200 μmol/L butyl hydroxy anisol for 1, 3 and 5 hours. Cells in the control group were treated with L-DMEM supplemented with 10% fetal calf serum. MAIN OUTCOME MEASURES： Morphologic changes of differentiated cells; Phenotype of neural cells was identified by immunofluorescence. RESULTS： After 24 hours of preinduction, cells changed from spindle to polygonal or irregular, with many short rod-shape processes and 2-3 nucleoli. Whirlpool shape growth disappeared, and was disordered. After 1-3 hours of induction, cells gradually became round, with significant cell contraction, strong refraction, bipolar or pluripolar processes connected each other into a net. After 5 hours of induction, grades one and two branches were detected. After 1-hour induction, some of the BMSC-derived cells expressed nestin. After 3-hour induction, a large number of cells continuously expressed nestin, and some of them expressed neuron specific enolase （NSE） and β-tubulin. The expression of NSE and β -tuhulin increased, but the expressiou of nestin remarkably decreased after 5 hours. No significant changes were detected in the control group, and cells were negative for nestin. CONCLUSION： Preinduction was performed using basic fibroblast growth factor, which could promote the differentiation from BMSCs into nerve precursor cells or neurons. The expression of neuroprotein molecule was coincided with the development of neural cells.