组织型转谷氨酰胺酶2在鼻黏膜间充质干细胞分化过程中的表达

Expression of tissue transgiutaminase 2 in differentiation of nasal mucosal mesenchymal stem cells

背景:外胚层间充质干细胞可在特定的外环境中定向分化为其他类型细胞,分化过程中一些特殊蛋白的表达可能直接影响其命运的方向。目的:探讨大鼠鼻黏膜间充质干细胞在成骨细胞分化和成神经样细胞分化过程中组织型转谷氨酰胺酶2的表达。方法:组织贴壁法分离培养并鉴定大鼠鼻黏膜间充质干细胞,分别进行成神经样细胞、成骨细胞诱导分化。在诱导不同时间点,采用免疫荧光检测神经相关蛋白GAP-43、TUBB3和骨相关蛋白OCN、COLⅠ的表达,免疫印迹法检测神经相关蛋白GAP-43、TUBB3,骨相关蛋白OCN、COLⅠ,神经营养因子NT-3、BDNF,细胞外基质FN、LN以及组织型转谷氨酰胺酶2的表达水平。结果与结论:①成神经诱导3 d,免疫荧光法观察可见神经元细胞特征性蛋白GAP-43和TUBB3呈阳性表达;成骨诱导7 d,免疫荧光法观察可见成骨细胞特征性蛋白COLⅠ和OCN呈阳性表达;②成神经诱导培养3,5,7 d,免疫印迹法检测神经细胞相关蛋白GAP-43、TUBB3和组织型转谷氨酰胺酶2表达水平随诱导时间延长而增高;成骨诱导7,14 d,免疫印迹法检测成骨细胞相关蛋白COLⅠ、OCN以及组织型转谷氨酰胺酶2水平随诱导时间延长而增高;③成神经诱导7 d,免疫印迹法检测神经营养因子NT-3、BDNF和组织型转谷氨酰胺酶2水平高于未诱导对照组;成骨诱导14 d,免疫印迹法检测细胞外基质FN、LN和组织型转谷氨酰胺酶2水平高于未诱导对照组;④结果表明,组织型转谷氨酰胺酶2可能参与和调控鼻黏膜间充质干细胞向神经样细胞和成骨细胞分化过程。

BACKGROUND:Ectodermal mesenchymal stem cells can differentiate into other types of cells in a specific external environment.The expression of some specific proteins during differentiation may directly affect the direction of their fate.OBJECTIVE:To investigate the expression of tissue transgiutaminase 2 in rat nasal mucosal mesenchymal stem cells during osteoblast differentiation and neuron-like differentiation.METHODS:Rat nasal mucosal mesenchymal stem cells were isolated and cultured by tissue adherence method,and differentiated into neuroblast-like cells and osteoblasts.Immunofluorescence was used to detect the expression of neuro-related proteins GAP-43,TUBB3 and bone-associated proteins OCN and COLI at different time points.Western blot assay was used to detect neuro-related proteins GAP-43,TUBB3,bone-associated protein OCN and COLI,neurotrophic factor NT-3,BDNF,extracellular matrix FN,LN,and tissue transgiutaminase 2.RESULTS AND CONCLUSION:(1)After 3 days of neurogenic induction,immunofluorescence showed that GAP-43 and TUBB3 expressed positively.At 7 days after osteogenic induction,COLI and OCN were positive under immunofluorescence staining.(2)At 3,5,7 days after neurogenic induction,the expression levels of nerve cell-associated proteins GAP-43,TUBB3 and tissue transgiutaminase 2 were increased with time detected by western blot assay.At 7 and 14 days after osteogenic induction,the levels of osteoblast-associated proteins COLI,OCN and tissue transgiutaminase 2 were increased with time.(3)At 7 days after neurogenic induction,western blot assay showed that the levels of NT-3,BDNF and tissue transgiutaminase 2 were higher than those in the non-induction group.At 14 days after osteogenic induction,the expression levels of FN,LN and tissue transgiutaminase 2 in the extracellular matrix were higher than those in the non-induction group.(4)To conclude,tissue transgiutaminase 2 may regulate the differentiation of nasal mucosal mesenchymal stem cells into neuron-like cells and osteoblasts.