中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (6): 973-978.doi: 10.3969/j.issn.1673-8225.2010.06.005

• 干细胞培养与分化 • 上一篇    下一篇

人羊膜上皮细胞分泌神经营养因子诱导人脐血间充质干细胞向神经元样细胞的分化：可能性验证

张晓明1，孙海梅1，杨 慧2，季凤清1   

  1. 首都医科大学，  1组织胚胎学系， 2神经生物学系，北京市 100069
  • 出版日期:2010-02-05 发布日期:2010-02-05
  • 通讯作者: 季凤清，博士，副教授，首都医科大学组织胚胎学系，北京市 100069 jfq@ccmu.edu.cn
  • 作者简介:张晓明★，女，1979年生，江苏省阜宁县人，汉族，首都医科大学在读硕士，主要从事脐血间充质干细胞方面的研究。 zxmailb@sohu.com
  • 基金资助:

    北京市教委科技发展计划项目(KM200610025005)；
    北京市自然科学基金(7082017)。

Human amniotic epithelial cells-secreted neurotrophic factors induces the differentiation of human umbilical cord blood mesenchymal stem cells into neuron-like cells: Possibility verification

Zhang Xiao-ming1, Sun Hai-mei1, Yang Hui2, Ji Feng-qing1   

  1. 1Department of Histology and Embryology,
    2Institute for Neuroscience, Capital Medical University, Beijing   100069, China
  • Online:2010-02-05 Published:2010-02-05
  • Contact: Ji Feng-qing, Doctor, Associate professor, Department of Histology and Embryology, Capital Medical University, Beijing 100069, China jfq@ ccmu. edu.cn
  • About author:Zhang Xiao-ming★, Studying for master's degree, Department of Histology and Embryology, Capital Medical University, Beijing 100069, China zxmailb@sohu.com
  • Supported by:

    the Scientific Research Fund Projects of Beijing Educational Council, No. KM200610025005*;
    the Beijing Municipal Natural Science Foundation, No. 7082017*

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摘要：

背景：课题组前期实验已证实人羊膜上皮细胞条件培养液可以诱导人脐血间充质干细胞分化为多巴胺能神经元样细胞，在此过程中人羊膜上皮细胞分泌的神经营养因子及其受体可能起到了重要作用。

目的：探讨人羊膜上皮细胞分泌的神经营养因子对人脐血间充质干细胞神经分化的作用。

方法：将P1代人脐血间充质干细胞按2×108 L-1密度接种，分为3组：对照组加入HG-DMEM培养基；诱导组加入人羊膜上皮细胞条件培养液；阻断剂组预先加入阻断剂K252a工作液，36 ℃孵育40 min后更换为羊膜上皮细胞条件培养液。免疫荧光化学检测诱导后人脐血间充质干细胞神经元特异性烯醇化酶及多巴胺转运体的表达，实时定量PCR法检测诱导后人脐血间充质干细胞中神经元特异性烯醇化酶、多巴胺转运体及酪氨酸羟化酶的表达。

结果与结论：人羊膜上清中有神经生长因子和脑源性神经营养因子的表达，且P1代人脐血间充质干细胞表达神经营养因子高黏附性受体Trka及Trkb。诱导48 h后与对照组比较，诱导组及阻断剂组神经元特异性烯醇化酶、多巴胺转运体阳性细胞数均明显增加(P < 0.05)，且诱导组阳性细胞数最多(P < 0.05)。诱导组、阻断剂组神经元特异性烯醇化酶、多巴胺转运体及酪氨酸羟化酶mRNA含量均显著高于对照组(P < 0.01)，且诱导组各基因mRNA含量明显高于阻断剂组(P < 0.01)。结果证实人羊膜上皮细胞分泌的神经营养因子对人脐血间充质干细胞的神经分化有重要作用，其促神经分化作用是通过酪氨酸激酶受体介导的。

关键词: 神经营养因子, 人脐血间充质干细胞, 羊膜上皮细胞, 诱导, 阻断, 干细胞

Abstract:

BACKGROUND: Group pre-test has confirmed that amnion endothelial cell conditioned medium can induce human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells. In this process, neurotrophic factors and their receptors may play an important role.

OBJECTIVE: To study the function of neurotrophic factors secreted by amniotic epithelial cells in the differentiation of human umbilical cord blood mesenchymal stem cells into neurons.

METHODS: P1 human umbilical cord blood mesenchymal stem cells at 2×108 /L were incubated and assigned to 3 group. Control group was added with HG-DMEM medium. Induction group received human amniotic epithelial cell medium. Blocking agent group underwent blocking agent K252a fluid, and the incubated was conducted at 36 ℃ for 40 minutes, and then amniotic epithelial cell medium was added. Immunofluorescence chemistry was used to determine neuron specific enolase and dopamine transporter expression in human umbilical cord blood mesenchymal stem cells. Real-time quantitative PCR was employed to detect neuron specific enolase, dopamine transporter and tyrosine hydroxylase expression in human umbilical cord blood mesenchymal stem cells.

RESULTS AND CONCLUSION: Nerve growth factor and brain-derived neurotrophic factor were observed in human amniotic supernatant. P1 human umbilical cord blood mesenchymal stem cells expressed Trka and Trkb. Forty-eight hours following induction, compared with the control group, positive expression of neuron specific enolase and dopamine transporter was significantly increased in the induction and blocking agent groups (P < 0.05), especially in the induction group (P < 0.05). Neuron specific enolase, dopamine transporter and tyrosine hydroxylase mRNA levels were significantly greater in the induction and blocking agent groups compared with the control group (P < 0.01), and each gene mRNA levels were significantly greater in the induction group than in the blocking agent group (P < 0.01). Results verified that neurotrophic factor in the human amniotic epithelial cells plays important effects on differentiation of human umbilical cord blood mesenchymal stem cells into neurons. The promotion effects are mediated by activating Trk receptor.

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