中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (8): 1462-1469.doi: 10.4103/1673-5374.253532

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

骨髓间充质干细胞由许旺细胞条件培养基诱导分化为神经元样细胞中miRNA变化的分析

  

  • 出版日期:2019-08-15 发布日期:2019-08-15
  • 基金资助:

    国家自然科学基金(81330042，81620108018，81702147)

MicroRNA changes of bone marrow-derived mesenchymal stem cells differentiated into neuronal-like cells by Schwann cell-conditioned medium

Zhi-Jian Wei 1 , Bao-You Fan 1 , Yang Liu 1 , Han Ding 1 , Hao-Shuai Tang 1 , Da-Yu Pan 1 , Jia-Xiao Shi 1 , Peng-Yuan Zheng 1 , Hong-Yu Shi 1 , Heng Wu 2 , Ang Li 3 , Shi-Qing Feng 1   

  1. 1 Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin, China
    2 Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin, China
    3 Department of Orthopedics, Henan Provincial People’s Hospital, Zhengzhou, Henan Province, China
  • Online:2019-08-15 Published:2019-08-15
  • Contact: Shi-Qing Feng, MD, sqfeng@tmu.edu.cn.
  • Supported by:

    The present study was supported by the National Natural Science Foundation of China, No. 81330042, 81620108018 (both to SQF), and 81702147 (to ZJW).

PDF

114

摘要：

研究已证实在许旺细胞诱导条件下，骨髓间充质干细胞能够向神经元方向分化，但分化过程中的关键miRNA及相关通路还不清楚。为此，实验筛选和鉴定许旺细胞条件培养基诱导骨髓间充质干细胞向神经元样细胞分化过程中差异表达的microRNA，并探讨microRNA靶基因和相关信号通路。从Wistar大鼠股骨和胫骨中分离原代骨髓间充质干细胞，从双侧隐神经中分离原代许旺细胞，用正常培养基与许旺细胞条件培养基分别培养，设为对照组和许旺细胞条件培养基诱导组。通过延时成像观察到许旺细胞条件培养基诱导骨髓间充质干细胞向神经元样细胞分化。(1)许旺细胞条件培养基诱导组中的骨髓间充质干细胞的细胞形态变为具有神经突的神经元样细胞。(2) qRT-PCR检测显示，许旺细胞条件培养基诱导组骨髓间充质干细胞中Nestin mRNA表达从培养1-3 d开始上调，到3-7 d开始下调，与对照组相比微管相关蛋白2 mRNA表达从培养1-7 d逐渐增加。(3)诱导7 d后，MicroRNA array用于分析差异microRNA的表达，两组相比共发现83个表达显著不同miRNA。(4) GO分析表明，这些差异miRNA的靶基因富集于神经元发育、轴突发生的调节并对细胞增殖起正性调控作用。(5)KEGG分析显示，Hippo，Wnt，转化生长因子β和Hedgehog信号通路是骨髓间充质干细胞向神经分化相关的潜在作用通路。(6) 结果证实，实验成功对骨髓间充质干细胞在许旺细胞诱导下分化神经元样细胞进行了miRNAs分析，进一步分析了骨髓间充质干细胞神经分化中的关键miRNA和通路。

orcid: 0000-0001-9437-7674 (Shi-Qing Feng)

关键词: microRNA分析, 骨髓间充质干细胞, 许旺细胞, 神经元样细胞, 神经方向分化, Gene Ontology (GO) 分析, Hippo 信号通路, Wnt 信号通路, 转化生长因子&beta, 信号通路, Hedgehog 信号通路, 神经再生

Abstract:

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone mar¬row-derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cul¬tured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was ob¬served by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesenchymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis identified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection devel¬opment, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathways were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Com¬mittee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

Key words: nerve regeneration, microRNA analysis, bone marrow-derived mesenchymal stem cells, Schwann cells, neuronal-like cells, neuronal differentiation, Gene Ontology analysis, Hippo signaling pathway, Wnt signaling pathway, transforming growth factor-beta signaling pathway, Hedgehog signaling pathway, neural regeneration