解剖学报

解剖学报 ›› 2023, Vol. 54 ›› Issue (3): 305-312.doi: 10.16098/j.issn.0529-1356.2023.03.008

• 细胞和分子生物学 • 上一篇    下一篇

低氧低糖及血清剥夺联合处理抑制Nrf2信号通路诱发大鼠骨髓间充质干细胞氧化应激和凋亡

谢秋敏1 孙艳婷1 许皓2 刘蕙文1 易勤1 谭彬1 田杰3 朱静1*
  

  1. 1.重庆医科大学附属儿童医院儿研所，国家儿童健康与疾病临床医学研究中心，儿童发育疾病研究教育部重点实验室，儿科学重庆市重点实验室，重庆400014;    2.重庆医科大学附属儿童医院检验科，重庆400014; 3.重庆医科大学附属儿童医院心血管科（内科），重庆400014
  • 收稿日期:2022-03-04 修回日期:2022-05-28 出版日期:2023-06-06 发布日期:2023-06-06
  • 通讯作者: 朱静 E-mail:zhujing310@yahoo.com.cn
  • 基金资助:
    Islet-1 诱导MSCs特化心肌细胞的电生理构建与能量代谢重塑调控

Glucose and serum deprivation under hypoxia treatment inducing oxidative stress and apoptosis in rat bone marrow mesenchymal stem cells through inhibition of Nrf2 signaling pathway

XIE  Qiu-min1  SUN Yan-ting1  XU Hao2  LIU Hui-wen1  YI Qin1  TAN Bin1  TIAN Jie3  ZHU Jing1*   

  1. 1. Department of Pediatric Research Institute, Children’s Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing400014, China;   2. Department of Clinical Laboratory, Children’s Hospital of Chongqing Medical University, Chongqing400014, China;   3. Department of Cardiovascular (Internal Medicine), Children’s Hospital of Chongqing Medical University, Chongqing400014, China
  • Received:2022-03-04 Revised:2022-05-28 Online:2023-06-06 Published:2023-06-06
  • Contact: ZHU Jing E-mail:zhujing310@yahoo.com.cn

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摘要：

目的 探讨低氧低糖血清剥夺（GSDH）处理对大鼠骨髓间充质干细胞（BMSCs）氧化应激及凋亡的影响。  方法 在体外对提取纯化后的原代BMSCs利用低氧（1% O2）、低糖（1.0g/L）及血清剥夺联合处理建立BMSCs细胞损伤模型。采用集落形成实验、细胞周期测定以及CCK-8实验检测细胞增殖能力；划痕实验和Transwell实验检测细胞迁移能力；通过细胞凋亡试剂（AnnexinV-FITC/PI）、线粒体膜电位检测试剂（JC-1）染色和线粒体荧光探针（Mito-Trancker）检测细胞凋亡；细胞活性氧簇（ROS）和钙离子（Fluo-4AM）检测细胞氧化应激水平；Western blotting检测抗氧化应激相关分子谷胱甘肽过氧化物酶4（GPX4）等以及核因子红细胞系2相关因子2（Nrf2）信号通路中关键分子的蛋白表达水平。  结果 大鼠原代BMSCs表面标志物高表达CD29、CD71，低表达CD45、CD34；GSDH处理抑制BMSCs细胞增殖（P<0.05）和迁移（P<0.05），增加ROS和钙离子水平（P<0.05），且抑制抗氧化应激相关分子GPX4等蛋白表达（P<0.01）；与空白对照组相比，GSDH处理后BMSCs凋亡显著升高（P<0.05）， 线粒体膜电位降低（P<0.01），网络化减少（P<0.01）。Nrf2信号通路中Nrf2蛋白及下游关键分子的蛋白表达水平均降低（P<0.05）。  结论 GSDH处理诱发的BMSCs氧化应激及进一步导致的凋亡损伤与Nrf2信号通路被抑制有关。

关键词: 低氧低糖血清剥夺, 氧化应激, 骨髓间充质干细胞, 免疫印迹法, 大鼠

Abstract:

Objective To investigate the effects of glucose and serum deprivation under hypoxia（GSDH）treatment on oxidative stress and apoptosis in rat bone marrow mesenchymal stem cells (BMSCs), so to provide an experimental support for improving the therapeutic efficacy of BMSCs.   Methods The cell injury model was established by hypoxia (1% O2), hypoglycemia (1.0 g/L) and serum deprivation in vitro with extracted and purified rat primary BMSCs. The proliferation ability of BMSCs was detected by colony formation assay, cell cycle assay and CCK-8 assay; the migration ability was observed by wound healing assay and Transwell assay; BMSCs apoptosis was detected by apoptosis assay kit(AnnexinV-FITC/PI), mitochondrial membrane potential assay kit(JC-1) staining and mitochondrial fluorescence probe(Mito-Tracker) staining; Reactive oxygen species (ROS) and calcium ion were used to detect the levels of cellular oxidative stress; Western blotting was conducted to measure the expression level of anti-oxidative stress-related protein glutathione peroxidase 4(GPX4) and the key protein expression of nuclear factor erthroid 2-related factor 2(Nrf2) pathway.   Results Rat primary BMSCs highly expressed CD29 and CD71 and lowly expressed CD45 and CD34; GSDH treatment inhibited cell proliferation (P<0.05) and migration (P<0.05)  of BMSCs, increased ROS and calcium ion levels (P<0.05), and suppressed the protein expression of anti-oxidative stress-related protein GPX4 and GCLC (P<0.01); compared with the blank control group, the apoptosis of BMSCs was significantly increased after GSDH treatment (P<0.05) and the mitochondrial membrane potential (P<0.01) and network size (P<0.01) was reduced. The expression levels of Nrf2 protein as well as the downstream key proteins in the Nrf2 signaling pathway were decreased (P<0.05).   Conclusion The oxidative stress and the further apoptotic damage induced by GSDH treatment in BMSCs are related to the inhibition of Nrf2 signaling pathway.

Key words: Glucose and serum deprivation under hypoxia, Oxidative stress, Bone marrow mesenchymal stem cell, Western blotting, Rat 

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