论文部分内容阅读
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 culture method of nasopharyngeal ectodermal stem cells (ECTO-MSCs) to investigate the stem cell stem cell differentiation potential. Methods: ECTO-MSCs were cultured and expanded in the nasal respiratory mucosa of rats. ECTO-MSCs were cultured and expanded in vitro. Immunofluorescence staining was performed using antibodies against stem cell marker protein nestin, CD133, CD44 and vimentin. (Including dexamethasone, vitamin C and β-glycerophosphate) and neurobasal culture medium (B27, brain-derived nerve growth factor, all-trans Victoria A acid, sonic hedgehog factor) induced ECTO-MSCs to differentiate into osteoblasts and neurons; histochemical staining and immunofluorescence staining were used to evaluate the differentiation of ECTO-MSCs. Results: After induction with osteogenic induction agent, ECTO-MSCs alkaline phosphatase activity was significantly enhanced, forming a large number of alizarin red staining-positive calcium nodules on the cell surface. After induced by neural induction medium, ECTO-MSCs Changes to neuron-like morphology, high expression of neuronal cell marker protein NF-200, cells issued slender protrusions and connected to each other into a neural network. CONCLUSION: ECTO-MSCs are widespread in the nasal respiratory mucosa of rats and have multipotential differentiation potential. They can be used as seed cells for autograft to repair bone and nerve tissue injury.