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背景:Wnt 信号通路是细胞增殖分化的关键调控环节,但与骨髓间充质干细胞神经分化的联系并不十分明确。目的:寻找促进骨髓间充质干细胞向神经元样细胞分化的 Wnt 信号分子。方法:首先体外分离培养大鼠骨髓间充质干细胞并传代,行形态学观察,并以流式细胞学方法检测细胞表型 CD44,CD9,CD34和 CD45。采用碱性成纤维细胞生长因子分别联合 Wnt3a 或 Wnt5a 的方案诱导分化,应用免疫组化和反转录-聚合酶链反应方法比较 Wnt3a 和 Wnt5a 对骨髓间充质干细胞向神经元样细胞分化的影响。结果与结论:骨髓间充质干细胞为长梭形,CD9,CD44 高表达,CD34,CD45 低表达。Wnt3a 诱导组的巢蛋白和神经元特异烯醇化酶呈阳性,而胶质纤维酸性蛋白无明显表达,诱导后细胞的活力良好。Wnt5a 诱导组巢蛋白呈弱阳性表达,而神经元特异烯醇化酶及胶质纤维酸性蛋白阴性。反转录-聚合酶链反应结果显示,Wnt3a 诱导组巢蛋白在诱导前后均有表达,神经元特异烯醇化酶在诱导后 5 d 可见明显的扩增条带,10 d 后更加明显。胶质纤维酸性蛋白在诱导 10 d 后出现较弱的扩增条带。Wnt5a 组、对照组骨髓间充质干细胞在诱导后 10 d 巢蛋白有微弱表达,神经元特异烯醇化酶和胶质纤维酸性蛋白几乎无表达。提示 Wnt3a 分子能够促进体外培养的骨髓间充质干细胞向神经元样细胞分化。
BACKGROUND: Wnt signaling pathway is a key regulatory element of cell proliferation and differentiation. However, the relationship between Wnt signaling and neural differentiation of bone marrow mesenchymal stem cells is not clear. OBJECTIVE: To find Wnt signaling molecules that promote the differentiation of bone marrow mesenchymal stem cells into neuron-like cells. Methods: Rat bone marrow mesenchymal stem cells were isolated and cultured in vitro. Morphological observation was performed. The phenotypes of CD44, CD9, CD34 and CD45 were detected by flow cytometry. Bone marrow mesenchymal stem cells differentiate into neuron-like cells by using Wnt3a or Wnt5a in combination with Wnt3a or Wnt5a respectively. Immunohistochemistry and reverse transcription-polymerase chain reaction . RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells were long fusiform, high expression of CD9 and CD44, low expression of CD34 and CD45. Nestin and neuron-specific enolase in Wnt3a-induced group were positive, while glial fibrillary acidic protein was not expressed, and the viability of cells after induction was good. Wnt5a-induced nestin expression was weakly positive, while neuron-specific enolase and glial fibrillary acidic protein were negative. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the expression of nestin in Wnt3a-induced group was both before and after induction, and the neuron-specific enolase showed obvious amplification band at 5 days after induction, especially after 10 days. Glial fibrillary acidic protein appeared weak amplification bands after 10 days of induction. The Wnt5a group and the control group showed weak expression of nestin at 10 days after induction, and almost no expression of neuron specific enolase and glial fibrillary acidic protein. These results suggest that Wnt3a can promote the differentiation of BMSCs into neuron-like cells in vitro.