脂肪组织源性干细胞分步诱导分化为神经元样细胞  

A two-step protocol for induction of rat adipose tissue-derived stem cells into neuron-like cells

作　　者：陈盼盼[1] 张丽华[2] 董为人[1] 刘杰明[3] 张易[1] 乔伟光[1] 陈英华[1] 赵姝 郭家松[1] 

机构地区：[1]南方医科大学基础医学院组织学与胚胎学教研室,广东广州510515 [2]佛山市科学技术学院基础医学院,广东佛山528305 [3]南方医科大学附属顺德区人民医院神经外科,广东顺德528300

出　　处：《南方医科大学学报》2011年第3期512-517,共6页Journal of Southern Medical University

基　　金：广东省高校优秀中青年人才基金(C1010086);佛山市科技攻关项目(201008063);佛山市卫生局医学科研立项(2010177)

摘　　要：目的探讨脂肪组织源性干细胞(ADSCs)诱导分化为神经元样细胞的可行性。方法 ADSCs分离自雄性SD大鼠附睾脂肪垫,差速贴壁法纯化ADSCs,传至第3代培养细胞行流式细胞术行表型鉴定(CD106,CD11b,CD45,CD90,CD29,CD49d),并进行成脂诱导。取鉴定后的ADSCs用分步诱导法,即先用含10 ng/ml碱性成纤维细胞生长因子(bFGF)、20ng/ml表皮生长因子(EGF)、2%B27的DMEM/F12培养基I培养,诱导其向神经干细胞转分化,再用含10 ng/ml胶质细胞源性神经营养因子(GDNF),10 ng/ml脑源性神经营养因子(BDNF),1μmol/L维甲酸(RA)的培养基II诱导分化为神经元样细胞(NLCs),免疫细胞化学法鉴定细胞表面标记物巢蛋白(nestin),神经细胞特异性标志微管相关蛋白(MAP2),神经元核蛋白(NeuN)。结果分离纯化的ADSCs CD90、CD29表达强阳性;成脂诱导分化后细胞油红O染色阳性;ADSCs经培养基I培养7 d后聚集成球样细胞团悬浮于培养液中,表达nestin;继而用培养基II诱导分化4 h后细胞球逐渐贴壁,球周边少数细胞向外发出突起,形似神经元,MAP2、NeuN均呈阳性。结论特定条件下,ADSCs在体外可被逐步诱导分化为NLCs。Objective To investigate the differentiation potential of rat adipose tissue-derived cells（ADSCs） into neuron-like cells in vitro using a two-step induction protocol.Methods ADSCs isolated from the epididymal fat pads in male SD rats by means of differential attachment were cultured in vitro and subjected to adipogenic induction.After flow cytometric identification of the cell surface antigens CD106,CD11b,CD45,CD49d,CD90 and CD29,the third-passage ADSCs were induced to transdifferentiate into neural stem cell（NSC）-like cells in DMEM/F12 medium containing 10 ng/ml basic fibroblast growth factor（bFGF）,20 ng/ml epidermal growth factor（EGF） and 2% B27.The resultant NSC-like cells were then induced to differentiate into neuron-like cells in the neurobasal medium containing 10 ng/ml brain-derived neurotrophic factor（BDNF）,10 ng/ml glial cell line-derived neurotrophic factor（GDNF） and 1 μmol/L retinoic acid（RA）.Immunocytochemistry was employed to identify the expression of the cell surface markers nestin,MAP2 and NeuN.Results The isolated ADSCs were positive for CD90 and CD29,and oil red O staining of the induced adipose-like cells yielded positive results.The third-passage ADSCs induced for 7 days aggregated as floating cell spheres positive for NSC surface antigen nestin.Further induction in neurobasal medium for 4 h resulted in adhesion of the cell spheres and the formation of cell processes extending from some peripheral cells,suggesting a morphological resemblance to neurons.Most of the cells showed positivity for MAP2 and NeuN.Conclusion ADSCs can be induced to differentiate into neuron-like cells in vitro under appropriate conditions.

关 键 词：脂肪组织源性干细胞 分化 神经干细胞 神经元 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]