成人鼻黏膜间充质干细胞的体外培养和多向诱导分化

Culture and induced multilineage differentiation of mesenchymal stem cells derived from human nasal mucosa

目的:观察成人鼻腔呼吸黏膜内间充质干细胞(MSCs)的分布特征,建立鼻黏膜MSCs的体外培养方法,探讨该细胞向神经细胞及成骨细胞分化的潜能。方法:将成人鼻腔呼吸黏膜制成冷冻切片,用Nestin和Vi-mentin抗体进行免疫荧光染色,观察MSCs在鼻黏膜内的分布特征。对鼻黏膜细胞进行体外培养、传代,扩增出MSCs,用干细胞标志蛋白Nestin,CD133,Vimentin和Sall4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液(含地塞米松、维生素C和β-甘油磷酸钠)及神经诱导培养液(Neurobasal培养液含B27,ATRA,TSA)诱导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:Nestin和Vimentin免疫荧光染色阳性的MSCs主要存在于鼻黏膜的固有层内,通过体外培养、传代,可扩增出大量的MSCs,该细胞表达干细胞标志蛋白Nestin,CD133,Vimentin和Sall4;用成骨诱导剂诱导培养后,该细胞的碱性磷酸酶活性明显增强,茜素红染色显示细胞表面形成大量的钙结节;经神经诱导培养液诱导培养后,细胞生长出细长突起并互相连接成网,细胞高表达神经细胞标志蛋白NF-200和BM88。结论:成人鼻腔呼吸黏膜固有层内广泛存在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 mesenchymal stem cells（MSCs） derived from human nasal mucosa,and explore their stemness and differentiation potential.Method：Based on the observation of distribution of MSCs in human nasal mucosa,we cultured and proliferated MSCs in vitro and identified the expression of stem cell markers on them including Nestin,CD133,Vimentin and Sall4 with immunofluorescence.The MSCs were induced to differentiate to osteoblasts with medium containing dexamethasone,ascorbic acid and beta sodium glycerophosphate,and to neurons with Neurobasal medium containing B27,ATRA and TSA.Histochemistry and immunofluorescence were applied to evaluate the differentiation.Result：The nestin and vimentin immunofluorescence-positive MSCs existed extensively in human nasal mucosa.While the MSCs were cultured in the osteogenic-inducing medium,activities of alkaline phosphatase were increased significantly,and bone nodules were found on the surface of the osteoblasts by alizarin red staining.After the induction by neural-inducing medium,the MSCs adopted neuron like appearance with many slim protrusions interconnected as a network.The induced cells expressed neural markers NF-200 and BM88 strongly.Conclusion：The MSCs derived from human nasal mucosa are multipotent stem cells and can be utilized as seed cells to repair bone or neural injury.