中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (14): 2476-2480.doi: 10.3969/j.issn.1673-8225.2010.14.002 　

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

体外培养大鼠骨髓间充质干细胞向神经元样细胞分化： Wnt3a信号分子的诱导作用

阎文柱，秦书俭，刘学政，李德华   

  1. 辽宁医学院解剖学教研室，辽宁省锦州市  121001
  • 出版日期:2010-04-02 发布日期:2010-04-02
  • 作者简介:阎文柱★，男，1964年生，辽宁省朝阳市人，汉族，副教授， 2003年东北财经大学毕业，硕士，主要从事神经生物学研究。 1078607311@qq.com
  • 基金资助:

    辽宁省教育厅基金资助项目(2006T063，2008422)“RNAi沉默种子细胞抗原表达对修复骨缺损的影响”，“基因转染人脐带间充质干细胞移植治疗脑缺血大鼠的研究”。

Differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells in vitro: Induction of Wnt3a signaling molecules

Yan Wen-zhu, Qin Shu-jian, Liu Xue-zheng, Li De-hua   

  1. Department of Anatomy, Liaoning Medical University, Jinzhou  121001, Liaoning Province, China
  • Online:2010-04-02 Published:2010-04-02
  • About author:Yan Wen-zhu★, Master, Associate professor, Department of Anatomy, Liaoning Medical University, Jinzhou 121001, Liaoning Province, China 1078607311@qq.com
  • Supported by:

    a Grant from Department of Education of Liaoning Province, No. 2006T063*; 2008422*

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摘要：

背景：Wnt信号通路是细胞增殖分化的关键调控环节。已有证据显示此通路参与了对神经前体细胞增殖、分化以及决定细胞命运的调控。目前有关Wnt信号通路对间充质干细胞向神经元样细胞分化的作用还少见报道。
目的：寻找促进间充质干细胞向神经元样细胞分化的Wnt信号分子。
方法：采用密度梯度离心法在体外分离培养SD大鼠股骨间充质干细胞并培养。传代后通过形态学和流式细胞学检测细胞表面标志物CD29、CD44、CD34、CD45,筛选并鉴定培养细胞。采用神经营养因子碱性成纤维细胞生长因子分别联合Wnt3a和Wnt5a诱导方案，通过免疫组化和RT-PCR的方法比较Wnt3a、Wnt5a在间充质干细胞向神经元样细胞分化过程中的作用，以碱性成纤维细胞生长因子单独培养为对照。
结果与结论：间充质干细胞经培养、传代后，细胞贴壁生长，形态均一，呈长梭形，流式细胞学检测细胞表面标志物CD29、CD44高表达, CD34、CD45低表达。Wnt3a诱导后细胞巢蛋白、神经元特异性烯醇化酶呈阳性，胶质纤维酸性蛋白无明显表达，诱导后细胞的活力良好；Wnt5a诱导组及对照组巢蛋白呈弱阳性表达，神经元特异性烯醇化酶及胶质纤维酸性蛋白阴性。RT-PCR结果显示，Wnt3a诱导组巢蛋白在诱导前后均有表达，神经元特异性烯醇化酶在诱导后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后有比较弱的扩增条带出现。结果说明Wnt3a分子能够促进体外培养的间充质干细胞向神经元样细胞分化。

关键词: 神经元样细胞, 间充质干细胞, Wnt3a, 诱导, 大鼠, 干细胞

Abstract:

BACKGROUND: Wnt signaling pathway is the key to regulation of cell proliferation and differentiation. The evidence suggests that this pathway participates in the neural precursor cell proliferation, differentiation and determination of the regulation of cell fate. At present, the Wnt signaling pathway effects on the mesenchymal stem cells (MSCs) differentiated into neuron-like cells have not been reported.
OBJECTIVE: To seek Wnt signal molecule that promotes the differentiation of MSCs into neuron-like cells.
METHODS: MSCs were isolated from the femur of Sprague Dawley rats in vitro using the density gradient centrifugation, and then cultured. Following passage, cell surface markers CD29, CD44, CD34 and CD45 were measured using morphology and flow cytometry. These cells were selected and evaluated. Using neurotrophic factor and basic fibroblast growth factor combined with Wnt3a and Wnt5a induction scheme, effects of Wnt3a and Wnt5a during the differentiation of MSCs into neuron-like cells were compared utilizing immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Simple culture of basic fibroblast growth factor served as controls.
RESULTS AND CONCLUSION: Following culture and passage of MSCs, cells adhered to the wall, showing even spindle shape. Flow cytometry showed great expression of CD29 and CD44 and low expression of CD34 and CD45. Following Wnt3a induction, cells were positive for nestin, neuron specific enolase, but no significantly expressed glial fibrillary acidic protein. Following induction, cell viability was good. In the Wnt5a induction and control groups, weakly positive expression of nestin was found, but neuron specific enolase and glial fibrillary acidic protein were negative. RT-PCR results demonstrated that nestin expression was detected in the Wnt3a induction group before and after induction. Significant amplified bands for neuron specific enolase were detected at day 5 following induction, and became more significant at day 10. Weak bands for glial fibrillary acidic protein were visible at day 10 following induction. These indicated that Wnt3a can promote the differentiation of MSCs into neuron-like cells in vitro.

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