中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (3): 464-472.doi: 10.4103/1673-5374.266058

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

从诱导多能干细胞来源间充质干细胞转移线粒体可减轻缺氧缺血PC12细胞线粒体功能障碍

  

  • 出版日期:2020-03-15 发布日期:2020-05-26
  • 基金资助:

    国家自然科学基金项目（81671882，81471832），广东省自然科学基金项目（2016A030311039），广东省科技基金项目（2015A020212012，2017A020224012），广州市科学技术基金项目（201707010373）

Transfer of mitochondria from mesenchymal stem cells derived from induced pluripotent stem cells attenuates hypoxia-ischemia-induced mitochondrial dysfunction in PC12 cells

Yan Yang1, 2, Gen Ye1, 3, Yue-Lin Zhang1, Hai-Wei He1, Bao-Qi Yu4, Yi-Mei Hong1, Wei You1, Xin Li1   

  1. 1 Department of Emergency Medicine, Department of Emergency and Critical Care Medicine, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong Province, China
    2 Department of Emergency, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China
    3 Shantou University Medical College, Shantou, Guangdong Province, China
    4 Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Key Laboratory of
    Remodelling-related Cardiovascular Diseases, Ministry of Education, Beijing, China
  • Online:2020-03-15 Published:2020-05-26
  • Contact: Xin Li, MD, PhD,xlidoct@qq.com.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, No. 81671882, 81471832; the Natural Science Foundation of Guangdong Province of China, No. 2016A030311039; the Science and Technology Foundation of Guangdong Province of China, No. 2015A020212012, 2017A020224012; the Science and Technology Foundation of Guangzhou City of China, No. 201707010373 (all to XL).

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摘要：

神经元中线粒体功能障碍与缺氧缺血诱导的脑损伤有关。越来越多的证据表明，间充质干细胞已成为治疗缺氧缺血诱导的脑损伤的一种新型疗法，但其潜在机制尚未完阐明。(1)首先将1.5×105个PC12细胞与诱导多能干细胞来源的间充质干细胞以1：1比例共培养，然后用氯化钴干预24h。以Mito-sox测量PC12细胞的活性氧水平，以JC-1染色测量PC12细胞线粒体膜电位，以TUNEL染色分析PC12细胞的凋亡情况，以透射电镜评估PC12细胞中的线粒体形态，以流式细胞术测量线粒体从诱导多能干细胞来源的间充质干细胞向受损PC12细胞的转移情况。(2)以含有线粒体中含有绿色荧光蛋白的慢病毒感染诱导多能干细胞来源的间充质干细胞，然后将其与PC12细胞于Transwell小室中共同培养，用氯化钴干预24h。以检测PC12细胞ATP的水平。(3)结果显示，氯化钴诱导的PC12细胞损伤呈剂量依赖性。氯化钴干预后，与诱导多能干细胞来源的间充质干细胞共培养可显著减少PC12细胞的凋亡并恢复受损PC12细胞中线粒体膜电位。同时与诱导多能干细胞来源的间充质干细胞共培养，也能明显改善受损PC12细胞中线粒体肿胀、嵴消失和染色质深染的现象。而PC12细胞与诱导多能干细胞来源的间充质干细胞直接共培养后，2种细胞间可见隧道纳米管，且线粒体通过隧道纳米管在细胞间转移。氯化钴能显著提升线粒体转移的效率。而通过细胞松弛素D抑制隧道纳米管形成，可部分抑制诱导多能干细胞来源的间充质干细胞对氯化钴诱导的PC12细胞损伤的有益作用。(4)提示诱导多能干细胞来源的间充质干细胞对氯化钴诱导的PC12细胞损伤的改善作用，部分归因于线粒体的转移。

orcid: 0000-0003-0469-5121 (Xin Li)

关键词:

线粒体转移, 诱导多能干细胞, 间充质干细胞, 隧道纳米管, PC12细胞, 缺氧缺血, 脑损伤, 细胞凋亡, 线粒体膜电位, 神经再生

Abstract: Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury. Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology, the mechanisms are not fully understood. To address this issue, we first co-cultured 1.5 × 105 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1, and then intervened with cobalt chloride (CoCl2) for 24 hours. Reactive oxygen species in PC12 cells was measured by Mito-sox. Mitochondrial membrane potential (ΔΨm) in PC12 cells was determined by JC-1 staining. Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining. Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy. Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry. Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria. Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells. CoCl2-induced PC12 cell damage was dose-dependent. Co-culture with mesenchymal stem cells significantly reduced apoptosis and restored ΔΨm in the injured PC12 cells under CoCl2 challenge. Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling, the disappearance of cristae, and chromatin margination in the injured PC12 cells. After direct co-culture, mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells. The transfer efficiency was greatly enhanced in the presence of CoCl2. More importantly, inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury. Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.

Key words: apoptosis, brain injury, hypoxia-ischemia, induced pluripotent stem cells, mesenchymal stem cells, mitochondrial membrane potential, mitochondrial transfer, PC12 cells, tunneling nanotubes