人脐血间质干细胞向神经元样细胞分化的诱导因素  

Influential factors of human umbilical cord blood-derived mesenchymal stem cells differentiating into neuron-like cells

作　　者：赵林娜[1] 李建斌[1] 单泓[1] 段艳丽[1] 焦红亮[2] 宁晓琳 马会娟 杨朋辉 龚桂玲 

机构地区：[1]河南省红十字血液中心,河南省郑州市450053 [2]郑州大学第一附属医院神经外科,河南省郑州市450003 [3]深圳北科细胞工程研究所,广东省深圳市510075

出　　处：《中国组织工程研究与临床康复》2008年第21期4049-4053,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景：研究证明脐血间质干细胞可向神经元样细胞分化，要同时保证脐血间质干细胞的扩增能力与分化潜能，其培养和诱导分化条件的优化显得尤为重要。目的：分析筛选人脐血间质干细胞向神经元样细胞体外诱导分化过程的影响因素。设计、时间及地点：随机对照实验，于2006—08／2007-05在河南省红十字血液中心血液成分应用研究所完成。材料：脐血来自郑州市妇幼保健院正常足月分娩的胎儿，平均采血量95mL。重组人表皮生长因子、碱性成纤维细胞生长因子购自Sigma公司；分离细胞所用的四联袋为山东威高集团公司产品。方法：取采集6h内的脐血，采用四联袋密度梯度离心法分离脐血单个核细胞，以5×10^9L^-1接种于DMEM／F12培养基中。①细胞因子实验：单独细胞因子组加入20μg／LB27、10μg／L碱性成纤维细胞生长因子；细胞因子联合组在此基础上加入5μg／L重组人表皮生长因子。②红细胞混入量实验：裂解组加入红细胞裂解液1mL，裂解后红细胞混入量为（0．44±0．13）×10^8／份；少红细胞组红细胞混入量为（0．51±0．21）×10^8／份，多红细胞组红细胞混入量为（2．65±1．28^）×108／份。③首次换液时间实验：分别于接种后48h、7d首次换液。④诱导分化：将碱性成纤维细胞生长因子、重组人表皮生长因子共诱导7d的细胞爬片进行巢蛋白免疫组织化学染色。主要观察指标：观察不同因素对脐血间质干细胞增殖分化的影响。检测诱导分化后神经干细胞巢蛋白的表达。结果：培养7d后，表皮生长因子与碱性成纤维细胞生长因子联合诱导促细胞增殖的效果优于单独应用碱性成纤维细胞生长因子（t=2．880，P〈0．05）：红细胞裂解液组、多红细胞组贴壁细胞数明显低于少红细胞组（t=-7．332～7．550，P〈0.05），即混入的红细胞总量不大于10^8／份对细BACKGROUND： Studies have identified that, human umbilical cord blood-derived mesenchymal stem cells （HUCB-derived MSCs） may differentiate into neuron-like cells, and simultaneously requires to maintain the amplification ability and differentiating potency of HUCB-derived MSCs. It is extremely important to optimize the conditions of culture and differentiation. OBJECTIVE： To analyze and screen the factors inducing HUCB-derived MSCs to differentiate into neuron-like cells in vitro. DESIGN, TIME AND SETTING： A randomized control experiment was carded out in the Blood Components Application Institute of Red-cross Blood Center of Henan Province （Zhengzhou, Henan, China） from August 2006 to May 2007. MATERIALS： Umbilical cord blood was from the term-delivery fetus in Zhengzhou Hospital of Matemal and Child Health （China）, the average blood volume was 95 mL. Recombinant human epidermal growth factor （EGF） and basic fibroblast growth factor （bFGF） were purchased from Sigma Company （USA）; Quadruple blood bag for the isolation was produced by Shandong Weigao Group （China）. METHODS： Umbilical cord blood samples were collected within 6 hours, and umbilical blood mononuclear cells were separated with quadruple bags by means of density gradient centrifugation. Cytokine trial： single cytokine group was added with 20 μ g/L B27 and 10 μ g/L bFGF; combined cytokine group was added with 20 μ g/L B27, 10 μ g/L bFGF and 5 μ g/L recombinant human EGF.Erythrocyte interfusion trial： cell lysis group was added with 1 mL erythrocyte lysis liquid, and the quantity of lyzed erythrocyte was （0.44±0.13）× 10^8, while the quantity of few erythrocyte group and many erythrocyte group was （0.51 ±0.21）× 10^8 and （2.65± 1.28）× 10^8, respectively. First medium change trial： the first medium changing time was 48 hours and 7 days following the incubation. Induced differentiation： induced by bFGF and recombinant human EGF, cells were stained with nestin immunohistochemistry.

关 键 词：脐血间质干细胞 神经元样细胞 诱导分化 影响因素 

分 类 号：R394.2[医药卫生—医学遗传学]