中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2018, Vol. 13 ›› Issue (1): 145-153.doi: 10.4103/1673-5374.224383

• 原著:颅神经损伤修复保护与再生 • 上一篇    下一篇

低频脉冲电磁场预处理骨髓间充质干细胞促进挤压伤面神经的再生

  

  • 收稿日期:2017-12-02 出版日期:2018-01-15 发布日期:2018-01-15
  • 基金资助:

    韩国健康、福利部健康工业发展研究所基金

Low-frequency pulsed electromagnetic field pretreated bone marrow-derived mesenchymal stem cells promote the regeneration of crush-injured rat mental nerve

NaRi Seo1, 3, Sung-Ho Lee2, 3, Kyung Won Ju2, 3, JaeMan Woo2, BongJu Kim4, SoungMin Kim1, 2, Jeong Won Jahng3, Jong-Ho Lee1, 2, 3, 4   

  1. 1 Department of Oral and Maxillofacial Surgery, Graduate School of Dentistry, Seoul National University, Seoul, South Korea
    2 Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, Seoul, South Korea
    3 Dental Research Institute, Seoul National University, Seoul, South Korea
    4 Clinical Translational Research Center for Dental Science (CTRC), Seoul National University Dental Hospital, Seoul, South Korea
  • Received:2017-12-02 Online:2018-01-15 Published:2018-01-15
  • Contact: Jong-Ho Lee, D.D.S., M.S.D.,Ph.D., leejongh@snu.ac.kr.
  • Supported by:

    This study was supported by a grant of the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI15C1535).

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摘要：

基于骨髓间充质干细胞的细胞疗法已显示出促进损伤周围神经再生的作用。脉冲电磁场可促进骨髓间充质干细胞增殖和神经分化。低频脉冲电磁场可在无神经生长因子的条件下诱导骨髓间充质干细胞的神经分化。实验通过体外和体内实验两部分评估低频脉冲电磁场预处理对骨髓间充质干细胞增殖和生物功能的影响，以及低频脉冲电磁场预处理骨髓间充质干细胞（PMSCs）促进损伤周围神经再生作用。体外实验中，以定量DNA分析检测骨髓间充质干细胞增殖情况，以RT-PCR法检测骨髓间充质干细胞中许旺细胞标志物S100，星形胶质细胞标志物胶质纤维酸性蛋白，神经营养因子脑源性神经营养因子和神经生长因子mRNA表达。体内实验中，以钳夹法建立大鼠面神经挤压伤模型，然后随机给予其面神经损伤部位注射低频脉冲电磁场预处理的骨髓间充质干细胞，未预处理的骨髓间充质干细胞，或PBS，细胞浓度为1×106个。应用荧光显微镜观察注射到受损面神经中的DiI标记的骨髓间充质干细胞数量，以检测细胞存活情况。细胞注射后1和2周以Von Frey纤维丝测痛法评估受损面神经的功能恢复。细胞注射后2周，使用组织形态计量学分析和逆行标记三叉神经节（TG）评估轴突再生。体外实验结果显示，相较于未预处理的骨髓间充质干细胞组，预处理的骨髓间充质干细胞组干细胞增殖更明显，生长因子mRNA表达更高。体内实验结果显示，预处理的骨髓间充质干细胞组受损面神经功能恢复较未预处理的骨髓间充质干细胞组更快，髓鞘化轴突数量和密度更高，三叉神经节逆行标记阳性的神经元数目也更多。因此，低频脉冲电磁场预处理骨髓间充质干细胞是提高细胞疗法促进周围神经再生效果的有效工具。

orcid:0000-0002-8843-545X(Jong-Ho Lee)

关键词: 神经再生, 间充质干细胞, 低频脉冲电磁场, 周围神经损伤, 面神经挤压伤

Abstract:

Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to promote the regeneration of injured peripheral nerves. Pulsed electromagnetic field (PEMF) reportedly promotes the proliferation and neuronal differentiation of BMSCs. Low-frequency PEMF can induce the neuronal differentiation of BMSCs in the absence of nerve growth factors. This study was designed to investigate the effects of low-frequency PEMF pretreatment on the proliferation and function of BMSCs and the effects of low-frequency PEMF pre-treated BMSCs on the regeneration of injured peripheral nerve using in vitro and in vivo experiments.In in vitro experiments, quantitative DNA analysis was performed to determine the proliferation of BMSCs, and reverse transcription-polymerase chain reaction was performed to detect S100 (Schwann cell marker), glial fibrillary acidic protein (astrocyte marker), and brain-derived neurotrophic factor and nerve growth factor (neurotrophic factors) mRNA expression. In the in vivo experiments, rat models of crush-injured mental nerve established using clamp method were randomly injected with low-frequency PEMF pretreated BMSCs, unpretreated BMSCs or PBS at the injury site (1 × 106 cells). DiI-labeled BMSCs injected at the injury site were counted under the fluorescence microscope to determine cell survival. One or two weeks after cell injection, functional recovery of the injured nerve was assessed using the sensory test with von Frey filaments. Two weeks after cell injection, axonal regeneration was evaluated using histomorphometric analysis and retrograde labeling of trigeminal ganglion neurons. In vitro experiment results revealed that low-frequency PEMF pretreated BMSCs proliferated faster and had greater mRNA expression of growth factors than unpretreated BMSCs. In vivo experiment results revealed that compared with injection of unpretreated BMSCs, injection of low-frequency PEMF pretreated BMSCs led to higher myelinated axon count and axon density and more DiI-labeled neurons in the trigeminal ganglia, contributing to rapider functional recovery of injured mental nerve. These findings suggest that low-frequency PEMF pretreatment is a promising approach to enhance the efficacy of cell therapy for peripheral nerve injury repair.

Key words: nerve regeneration, mesenchymal stem cells, low-frequency pulsed electromagnetic field, peripheral nerve injury, crush-injured mental nerve