人胎盘间充质干细胞体外向胆碱能样神经元的分化  

Differentiation of human placenta-derived mesenchymal stem cells into cholinergic neuron-like cells in vitro

作　　者：井绪东[1] 何冬梅[1] 张洹[1] 陈丽[1] 

机构地区：[1]暨南大学医学院,血液病研究所,广东省广州市510632

出　　处：《中国组织工程研究与临床康复》2010年第32期5949-5951,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国务院侨办重点学科建设基金资助(51205002)~~

摘　　要：背景:胎盘中含有与骨髓中类似的间充质干细胞,也可能具有多向分化能力。目的:探讨人胎盘间充质干细胞体外能否定向诱导分化为胆碱能样神经元。方法:采用酶消化法分离培养人胎盘间充质干细胞,传至第4代,先用胎牛血清+碱性成纤维细胞生长因子诱导培养2d,然后分为3组:方案Ⅰ加入含碱性成纤维细胞生长因子、2-巯基乙醇、维甲酸、神经生长因子的DMEM/F12继续诱导19d;方案Ⅱ在其基础上,诱导液中另加入表皮生长因子、Heparin;阴性对照组仅加入含胎牛血清的DMEM/F12培养液,不添加任何诱导剂。结果与结论:胎盘组织消化后获得的贴壁细胞,经2周培养后逐渐形成扁平单层细胞,呈平行排列或漩涡样成簇生长,为梭形,传至第3代细胞形态较均一。第4代人胎盘间充质干细胞强表达CD44,CD29,不表达CD34,CD45,CD106及HLA-DR。人胎盘间充质干细胞经方案Ⅰ和方案Ⅱ诱导2周后,均可检测到nestin、chat mRNA的表达,且chat,Ache,nestin阳性率显著高于阴性对照组细胞(P<0.05)。与阴性对照组比较,经两种方案诱导的人胎盘间充质干细胞培养上清液中乙酰胆碱浓度均明显升高(P<0.05),且方案Ⅰ诱导组升高幅度明显大于方案Ⅱ诱导组(P<0.05)。提示联合多种生长因子分阶段进行诱导,人胎盘间充质干细胞可在体外分化为具有合成及释放降解乙酰胆碱能力的胆碱能样神经元细胞。BACKGROUND：Human placenta may contain similar mesenchymal stem cells （MSCs）,which can be differentiated into cholinergic neuron-like cells. OBJECTIVES：To explore the differentiation of MSCs from human placenta into cholinergic neuron-like cells in vitro. METHODS：MSCs from human placenta were isolated and purified by cell culture. The fourth passage of MSCs was incubated with fetal bovine serum＋basic fibroblast growth factor （bFGF） for 2 days,and divided into 3 groups：groupⅠ,MSCs were incubated with DMEM/F12 complemented with bFGF,2-mercaptoethanol,retinoic acid and nerve growth factor for 19 days. GroupⅡ,based on Group Ⅰ,epidermal growth factor and heparin were added into the culture system for 19 days. In the negative control group,cells were cultured by DMEM/F12 containing fetal bovine serum. RESULTS AND CONCLUSION：The adhesive cells gradually formed flat monolayer cells,parallel arranged or whirlpool grew,with uniform morphology after 2 passaged. The fourth passage of MSCs from human placenta was strongly positive for CD44,CD29,but negative for CD34,CD45,CD106 and HLA-DR. After 2 weeks of culture,nestin,chat mRNA could be expressed. The positive rate of nestin,Ache and chat mRNA was higher in the group Ⅰ and group Ⅱ than that of the negative control group （P 0.05）. Compared to the negative control group,the content of acetylcholine in supernate was obviously increased in the group Ⅰ and group Ⅱ （P 0.05）,especially more in the group Ⅰ （P 0.05）. The results demonstrated that MSCs from human placeta can differentiate into cholinergic neuron-like cells,which are able to synthesis,release and disintegrate acetylcholine.

关 键 词：生长因子 诱导 分化 胆碱能神经元 胎盘 间充质干细胞 干细胞 

分 类 号：R394.2[医药卫生—医学遗传学]