中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (10): 1711-1715.doi: 10.3969/j.issn.1673-8225.2010.10.001

• 干细胞培养与分化 •    下一篇

microRNA-125b在Flk1+脂肪源间充质干细胞向神经分化过程中的表达变化

王世华，边春景，黄  珊，赵春华   

  1. 中国医学科学院北京协和医学院基础所组织工程研究中心，北京市  100005
  • 出版日期:2010-03-05 发布日期:2010-03-05
  • 通讯作者: 赵春华，博士，博士生导师，中国医学科学院北京协和医学院基础所组织工程中心，北京市 100005 chunhua@public.tpt.tj.cn
  • 作者简介:王世华，女，1982年生，山东省青岛市人，汉族，北京协和医学院在读博士，主要从事成体干细胞的研究。 wshawp@163.com
  • 基金资助:

    国家高技术研究发展计划(八六三)基金(2006AA02A109；2006AA02A115)。

Differential expression of microRNA-125b in the neuronal differentiation of adipose-derived Flk1+ mesenchymal stem cells

Wang Shi-hua, Bian Chun-jing, Huang Shan, Zhao Chun-hua   

  1. Research Center of Tissue Engineering, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences＆Peking Union Medical College, Beijing  100005, China
  • Online:2010-03-05 Published:2010-03-05
  • Contact: Zhao Chun-hua, Doctor, Doctoral supervisor, Peking Union Medical College, Research Center of Tissue Engineering, Chinese Academy of Medical Sciences, Bejing 100005, China chunhua@public.tpt.tj.cn
  • About author:Wang Shi-hua, Studying for doctorate, Research Center of Tissue Engineering, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences＆Peking Union Medical College, Beijing 100005, China wshawp@ 163.com
  • Supported by:

    Special Foundation of the National Science and Technology Ministry of China (863 Program), No.2006AA02A109*, 2006AA02A115*

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摘要：

背景：间充质干细胞的分化受遗传和表观遗传因素的严格调控，越来越多的研究表明microRNA也在这一过程中发挥重要的调控作用。

目的：观察microRNA-125b在Flk1+间充质干细胞向神经细胞分化过程中的表达变化。

方法：成人脂肪样品取自15~35岁健康志愿者，贴壁培养法获得间充质干细胞，采用神经诱导培养基对第3代间充质干细胞进行诱导培养。在诱导第0，4，8，12天检测microRNA-125b的表达情况。应用microRNA芯片技术检测间充质干细胞在神经诱导前后microRNA-125b的表达差异并采用反转录-聚合酶链反应和Taqman real-time PCR的方法进行验证。检测inhibitor抑制microRNA-125b表达的有效性。

结果与结论：①实验成功诱导间充质干细胞向神经分化，诱导培养后12 d观察到细胞形态发生变化：大部分细胞胞体回缩，呈球形，并有轴突形成，免疫荧光检测结果显示，分化后的细胞表达神经元、星形胶质细胞特征性表面标志。②RT-PCR和Taqman real-time PCR显示神经诱导后microRNA-125b表达明显升高，与microRNA芯片结果一致。③inhibitor可以有效抑制microRNA-125b的表达。提示microRNA-125b可能在间充质干细胞成神经分化过程中起调控作用。

关键词: microRNA-125b, 脂肪源间充质干细胞, 神经分化, miRNA, miRNA芯片

Abstract:

BACKGROUND: The differentiation of mesenchymal stem cells (MSCs) is strictly regulated by both genetic and epigenetic factors. Emerging evidences have demonstrated that microRNA also plays an important role in this process.

OBJECTIVE: To explore the differential expression of microRNA-125b during neuronal differentiation of Flk1+ adipose-derived MSCs (AD-MSCs).

MATERIALS：The fat samples were provided by healthy female volunteers aged 15-35 years and recruited from the Plastic Surgery Hospital affiliated to the Chinese Academy of Medical Sciences.

METHODS: Adult adipose tissues were obtained from healthy volunteers with age of 15-35 years. Using adherence method, Flk1+ MSCs were obtained and the 3rd passage cells were taken in the experiment. Cultured in neuronal induction medium, these MSC were induced to differentiate towards neuronal lineage. The expression of microRNA-125b was examined at days 0, 4, 8 and 12. To explore its role in neuronal differentiation, we need to change its expression. RT-PCR and Taqman real-time PCR were carried out to explore the differentially expression of microRNA-125b during neuronal differentiation of AD-MSCs. The effect of inhibitor on the expression of microRNA-125b was detected.

RESULTS AND CONCLUTION: ①The Flk-1+ MSCs were successfully induced into neuronal differentiation and displayed typical morphological changes 12 days after induction: Most cells retracted their cytoplasm, forming spherical cell body and emitted cellular protrusions. RT-PCR and immunocytochemistry studies confirmed their phenotype with expression of known neuronal cell markers including neurofilament, glial fibrillary acidic protein. ②The expression of microRNA-125b was significant up-regulated during neuronal differentiation. Results of RT-PCR and Taqman real-time PCR were concordance with that of microRNA chip technology. ③Inhibitor could down-regulate microRNA-125b. The results implied that microRNA-125b may play an important role in neuronal differentiation.

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