中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (1): 19-23.doi: 10.3969/j.issn.1673-8225.2010.01.005

• 干细胞培养与分化 • 上一篇    下一篇

音猬因子诱导恒河猴骨髓间质干细胞向神经元样细胞的分化与维甲酸方案比较其信号分子表达变化的意义

宋 革1，2，张 阳3，刘炳乾2，郑炜炜1，孙雪融2   

  1. 1广东省计划生育科学技术研究所，广东省广州市 510600；
    2中山大学眼科学国家重点实验室，广东省广州市  510060；
    3解放军第四五八医院整形外科，广东省广州市  510602
  • 出版日期:2010-01-04 发布日期:2010-01-04
  • 作者简介:宋 革☆，女，1973年生，河南省郑州市人，汉族，2001年南方医科大学毕业，博士，副研究员，主要从事干细胞分化及信号传导方面的研究。 songgepp@126.com
  • 基金资助:

    国家自然科学基金（30500555），课题名称“恒河猴骨髓源性神经干细胞眼内移植分化迁移的信号机制研究”；
    广东省计生委重点课题（2008001）

Differentiation of rhesus monkey bone marrow mesenchymal stem cells into neuron-like cells induced by sonic hedgehog: Significance of its signal molecule expression changes compared with retinoic acid scheme

Song Ge1,2, Zhang Yang3, Liu Bing-qian2, Zheng Wei-wei1, Sun Xue-rong2   

  1. 1Guangdong Provincial Family Planning Research Institute of Science and Technology, Guangzhou   510600, Guangdong Province, China;
    2State Key Laboratory of Ophthalmology of Sun Yat-sen University, Guangzhou  510060, Guangdong Province, China;
    3Department of Plastic Surgery, the 458 Hospital of Chinese PLA, Guangzhou  510602, Guangdong Province, China
  • Online:2010-01-04 Published:2010-01-04
  • About author:Song Ge☆, Doctor, Associate investigator, Guangdong Provincial Family Planning Research Institute of Science and Technology, Guangzhou 510600, Guangdong Province, China; State Key Laboratory of Ophthalmology of Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China songgepp@126.com
  • Supported by:

    the National Natural Science Foundation of China, No.30500555*;
    the Family Planning Committee Program of Guangdong Province, No.2008001*

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381

被引次数

3

摘要：

背景：近来研究发现在神经发育过程中，神经干细胞的分化受到来自周围微环境中诸多调控因子的作用，其中音猬因子是神经胚胎发育过程中关键性的诱导信号，有望作为一种有效的诱导剂调节神经细胞的分化。

目的：探讨音猬因子诱导恒河猴骨髓间质干细胞向神经元样细胞分化过程中的信号分子表达变化。

方法：常规密度梯度离心法分离培养恒河猴骨髓间质干细胞，诱导组加入含FGF2、B27、胎牛血清的L-DMEM预诱导24 h撤离血清后，分别加入含0.5 μmol/L维甲酸或400 μmol/L音猬因子的DMEM诱导8 d，以未诱导的细胞作为对照组。经神经元烯醇化酶荧光标记后，流式细胞仪筛选阳性细胞，应用RT-PCR与Western-blot法检测音猬因子和维甲酸诱导的细胞膜受体、细胞内信号蛋白的变化。

结果与结论：音猬因子特异性膜受体Ptc、维甲酸特异性受体RARα、信号蛋白分子ptch1及Smad在正常细胞均有表达。经音猬因子诱导的细胞其Ptc表达上调，且随着诱导时间延长持续高表达，明显强于维甲酸诱导组及对照组(P < 0.01)；其细胞内ptch1蛋白分子表达与此趋势一致，但不能引起RARα表达上调。经维甲酸刺激的细胞在诱导过程中没有激活Ptc通路，诱导4 d后RARα表达出现小幅上调并持续至6 d，但这一结果差异不具有显著性意义，ptch1的表达无明显变化。经音猬因子、维甲酸诱导的细胞Smad分子表达均上调，但无明显差异。证实音猬因子诱导方案通过持续激活其特异性受体途径参与细胞诱导分化过程，而维甲酸诱导方案和音猬因子诱导方案之间没有显著受体途径交叉。

关键词: 音猬因子, 信号调控, 恒河猴, 骨髓间质干细胞, 干细胞

Abstract:

BACKGROUND: Present studies have demonstrated that during neural development, differentiation of neural stem cells (NSCs) was affected by various regulatory factors from surrounding microenvironment. Sonic hedgehog (Shh) is a key induction signal during neural fetal development, and can be an effective inductor to regulate differentiation of neural cells.
OBJECTIVE: To investigate the signal transduction pathway of SHH for differentiation of rhesus monkey bone marrow mesenchymal stem cells (BMSCs) into of neuron-like cells by sonic hedgehog factor.
METHODS: Rhesus monkey BMSCs were isolated and cultured by conventional density gradient centrifugation. BMSCs in the induction group were treated with L-DMEM containing FGF2, B27 and fetal bovine serum for preinduction of 24 hours, and then with DMEM supplemented with 0.5 μmol/L retinoic acid or 400 μmol/L SHH for 8 days. Non-induced cells served as control group. Following labeling with neuron enolase, positive cells were screened by flow cytometry. RT-PCR and Western-blot were used to detect SHH- and retinoic acid-induced cell membrane receptor and intracellular signal protein changes.
RESULTS AND CONCLUSION: SHH specific membrane receptor Ptc, retinoic acid specific receptor RARα, signal protein molecule ptch1 and Smad expressed in normal cells. Ptc expression upregulated in SHH-induced cells. High expression lasted for a long time with induction time, which was significantly stronger compared with the retinoic acid and control groups (P < 0.01). Intracellular ptch1 protein molecule expression showed similar tendency as this, but could not induce upregulation of RARα expression. During induction, retinoic acid-stimulated cells did not activate Ptc pathway. Four days following induction, RARα expression upregulated and lasted till 6 days, but there were no significant differences. No significant change in ptch1 expression was determined. SHH- and retinoic acid-induced cell Smad molecule expression upregulated, but no significant difference was determined. Results verified that SHH-induced scheme participated in cell induction and differentiation by persistently activating its specific receptors. However, there was no significant receptor pathway crossing between retinoic acid-induced and SHH-induced schemes.

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