人脱细胞羊膜对间充质干细胞向神经系细胞分化的影响  

Influence of human acellular amniotic membrane on the differentiation of mesenchymal stem cells into neural lineage cells

作　　者：胡炜[1] 关方霞[2] 杨波[1] 杜英[3] 马建[1] 李祥生[1] 胡祥 焦红亮[1] 李远[1] 

机构地区：[1]郑州大学第一附属医院神经外科,河南省郑州市450052 [2]郑州大学生物工程系,河南省郑州市450001 [3]郑州大学基础医学院微生物与免疫学教研室,河南省郑州市450052 [4]深圳市北科细胞工程研究所,广东省深圳市518000

出　　处：《中国组织工程研究与临床康复》2009年第14期2611-2617,共7页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：河南省医学科技创新人才工程项目(2005018)~~

摘　　要：背景:成体干细胞在细胞外基质加细胞因子作用下向神经系细胞分化的报道甚少。目的:根据干细胞巢原理,探讨羊膜间充质干细胞体外向神经系细胞诱导分化的条件,及其定向分化过程中神经蛋白的表达。设计、时间及地点:细胞形态学观察及蛋白分子水平检测,于2007-12/2008-04在郑州大学医学院实验中心完成。材料:产妇自愿捐献的羊膜由郑州大学第一附属医院产科提供。维甲酸、碱性成纤维细胞生长因子为Sigma公司产品。方法:体外分离培养、扩增羊膜间充质干细胞,调整细胞密度为4×107L-1,同时通过酶-化学法制作膜样基质。设立2组:诱导组羊膜间充质干细胞以基础培养基预诱导后,按4×107L-1接种在膜样基质上,并以神经基础培养基培养2d,然后替换为含10-3mmol/L维甲酸、20μg/Lβ-神经生长因子的DMEM/F12神经诱导培养基,继续培养24h,换基础培养基继续培养3d。对照组除不用膜样基质外,余步骤同诱导组。主要观察指标:细胞形态学变化,蛋白分子水平鉴定神经细胞表型。结果:诱导组接种到膜样细胞外基质24h可见细胞呈球形,紧贴膜样基质,胞体伸出突起,折光性强;第3天大部分细胞突起延伸并相互连接;4～6d突起形成网状结构且排列具有一定的方向性;对照组3d时见多个突起,4～6d细胞又恢复纤维细胞形态。预诱导后,两组巢蛋白、神经元特异性烯醇化酶表达明显增高,突触素、胶质纤维酸性蛋白表达无明显变化;诱导6d时与对照组比较,诱导组神经元特异性烯醇化酶表达无明显差异(P>0.05),突触素表达显著升高(P<0.01),巢蛋白、胶质纤维酸性蛋白表达显著降低(P<0.01)。结论:在膜样细胞外基质上,羊膜间充质干细胞可显示典型神经元特有的形态特征、表达标记。提示应用基质加细胞因子的诱导程序,可以从羊膜间充质干细胞高效获得神经元前体细胞,并抑制其向神经胶质细�BACKGROUND： The method of adult stem cell differentiation into neural cells by extracellular matrix （ECM） and cytokine have not been reported. OBJECTIVE： Based on theory of stem cell niche, to investigate the conditions for induction of amniotic-derived mesenchymal stem cells （ADMSCs） into neural lineage cells, and the neural markers＇ expression during differentiation. DESIGN, TIME AND SETTING： The cell morphology observation and protein molecular level detected were performed at the Experimental Center, Medical College, Zhengzhou University from December 2007 to April 2008. MATERIALS： Amnion donate by puerperant was obtained from the Department of Obstetrics, First Affiliated Hospital, Zhengzhou University. Basic fibroblast growth factor and retinoic （Sigma, USA） were used in this study. METHODS： ADMSCs were isolated from amnion in vitro. Membrane-like ECM was made by enzyme, chemical method, at a cell density of 4×10^7/L. Cells in the induction group were treated with basal medium for pre-induction. Cells at 4×10^7/L were seeded on the ECM and cultured by basal neuro-medium for two days, and then replaced it with DMEM/F12 neuro-inducted medium, supplemented with 10^-3 mmol/L retinoic acid and 20 μg/L β-nerve growth factor, and cultured for 24 hours, last cultured in the basal medium for 3 days. Cells in the control group were treated without ECM. MAIN OUTCOME MEASURES： Morphologic changes of differentiated cells, and phenotype of neural cells were measured. RESULTS： At hour 24, cells in the induction group were seeded on the ECM, round and sticked on ECM tightly, with processes stretched out, strong refraction. At day 3, most cell processes extended and connected each other. At days 4-6, processes connected each other into a net and arranged with directionality. At day 3, cells in the control group stretched out bipolar or pluripolar processes. At days 4-6, cells returned to fibroblast-like. After pre-induction, Nestin, neuron specific enolase expresstion of both groups increase

关 键 词：羊膜间充质干细胞 神经元 细胞外基质 分化 

分 类 号：R394.2[医药卫生—医学遗传学]