大鼠鼻黏膜外胚层间充质干细胞向成骨细胞和神经元的诱导分化  

Induced differentiation of ectomesenchymal stem cells derived from rat nasal mucosa to osteoblasts and neurons

作　　者：贺清华[1] 周月鹏[1] 陈谦[1] 刘锦波[1] 吴滢[1] 庄琴[1] 孙湘兰[1] 龚爱华[1] 姜平[1] 张志坚[1] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2011年第3期190-193,180,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30570981)

摘　　要：目的:建立鼻黏膜外胚层间充质干细胞(ectomesenchymal stem cells,ECTO-MSCs)的体外培养方法,探讨该细胞的干细胞特性(stemness)和分化潜能。方法:在观察ECTO-MSCs在大鼠鼻腔呼吸黏膜内分布特征的基础上,体外培养扩增ECTO-MSCs,并用干细胞标志蛋白巢蛋白、CD133、CD44、波形蛋白的抗体进行免疫荧光染色,鉴定该细胞的干细胞特性;分别用成骨诱导培养基(含地塞米松、维生素C和β-甘油磷酸钠)及神经诱导培养基(neurobasal培养液中加入B27、脑源性神经生长因子、全反式维A酸、音猬因子)诱导ECTO-MSCs向成骨细胞和神经细胞定向分化;用组织化学染色和免疫荧光染色方法,评价其诱导分化效果。结果:用成骨诱导剂诱导培养后,ECTO-MSCs碱性磷酸酶活性明显增强,在细胞表面形成大量的茜素红染色阳性的钙结节;用神经诱导培养基诱导培养后,ECTO-MSCs变化为神经细胞样形态,细胞高表达神经细胞标志蛋白NF-200,细胞发出细长突起并互相连接成神经网络。结论:大鼠鼻腔呼吸黏膜内广泛存在ECTO-MSCs,该细胞具有多向分化潜能,可作为种子细胞用于自体移植修复骨和神经组织损伤。Objective: To establish an in vitro method to culture ectomesenchymal stem cells(ECTO-MSCs) derived from nasal mucosa,and explore their stemness and differentiation potential.Methods: Based on the observation of distribution of ECTO-MSCs in rat nasal mucosa,we cultured and proliferated ECTO-MSCs in vitro and identified the expression of stem cell markers on them including nestin,CD133,CD44 and Vimentin with immunofluorescence.Besides,osteogenesis induced media,with ingredient of dexamethasone,ascorbic acid and beta sodium glycerophosphate,and neurons formation media,formed by adding B27,BDNF,all-trans-retinoicacid and sonic hedgehog into neurobasal media,were also applied to induce the differentiation of ECTO-MSCs to osteoblasts and neurons to confirm stemness.Immunohistology and immunofluorescence were applied to evaluate the differentiation.Results: While ECTO-MSCs were cultured in the osteogenesis-induced media,activities of alkaline phosphatase were increased significantly,and bone nodules were found on the surface of cells by alizarin red staining.After the induction by neuron forming media,ECTO-MSCs changed into neuron like appearance with many slim protrusions interconnected as a network.The induced cells expressed neural markers NF-200 strongly.Conclusion: ECTO-MSCs exist extensively in rat nasal mucosa,and are multipotent stem cells,therefore,can be utilized as seeder cells to repair bone or neural lesions.

关 键 词：鼻黏膜 外胚层间充质干细胞 诱导分化 成骨细胞 神经细胞 

分 类 号：R[医药卫生]