中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (1): 111-119.doi: 10.12307/2025.929

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

氟暴露对原代神经细胞内质网-线粒体钙转移及细胞凋亡的影响

卢永恒1，朱  双1，赵飞艳1，艾福军1，刘艳洁1，2，董阳婷3，官志忠1，2，3，魏  娜1，2   

  1. 1贵州医科大学临床医学院病理学教研室，贵州省贵阳市  550004；2贵州医科大学附属医院病理科，贵州省贵阳市  550004；3地方病与少数民族疾病(贵州医科大学)教育部重点实验室，贵州省贵阳市  550004
  • 收稿日期:2024-09-25 接受日期:2024-11-26 出版日期:2026-01-08 发布日期:2025-07-02
  • 通讯作者: 魏娜，博士，副教授，贵州医科大学附属医院病理科，贵州省贵阳市 550004；贵州医科大学病理学教研室，贵州省贵阳市 550004
  • 作者简介:卢永恒，男，1998年生，贵州省毕节市人，汉族，贵州医科大学病理学与病理生理学在读硕士，主要从事地方病病理与分子病理方面的研究。
  • 基金资助:
    国家自然科学基金项目(82360672)，项目负责人：魏娜；贵州医科大学自然科学基金培育项目(TJ23114)，项目负责人：魏娜；贵州省科技计划项目(黔科合基础-ZK[2022]一般439)，项目负责人：魏娜；贵州省科技计划项目(黔科合基础-ZK[2022]一般342)，项目负责人：董阳婷

Effect of fluoride exposure on endoplasmic reticulum-mitochondrial calcium transfer and apoptosis in primary nerve cells

Lu Yongheng1, Zhu Shuang1, Zhao Feiyan1, Ai Fujun1, Liu Yanjie1, 2, Dong Yangting3, Guan Zhizhong1, 2, 3, Wei Na1, 2   

  1. 1Pathology Teaching and Research Section, Clinical Medical College, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 2Department of Pathology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 3Key Laboratory of Endemic and Ethnic Minority Diseases (Guizhou Medical University) of Ministry of Education, Guiyang 550004, Guizhou Province, China
  • Received:2024-09-25 Accepted:2024-11-26 Online:2026-01-08 Published:2025-07-02
  • Contact: Wei Na, MD, Associate professor, Pathology Teaching and Research Section, Clinical Medical College, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; Department of Pathology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:Lu Yongheng, Master candidate, Pathology Teaching and Research Section, Clinical Medical College, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82360672 (to WN); Natural Science Foundation of Guizhou Medical University, No. TJ23114 (to WN); Guizhou Provincial Science and Technology Plan Project, No. ZK[2022]439 (to WN); Guizhou Provincial Science and Technology Plan Project, No. ZK[2022]342 (to DYT)

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摘要：

文题释义：

内质网-线粒体钙转运：是指Ca2+由内质网到线粒体之间的转运过程，这一过程在细胞内钙信号转导、能量代谢、细胞生存和死亡等多种生理和病理过程中发挥着重要作用。
线粒体相关内质网膜：是细胞内一种特殊结构，主要是线粒体和内质网相互靠近但不融合的区域，在细胞中涉及Ca2+稳态、氧化应激、脂质代谢和细胞凋亡等多种生理过程。

摘要
背景：前期研究发现持续过量氟暴露导致的神经细胞受损与Ca2+超载有关，但Ca2+在细胞内各钙库之间的流动转换和细胞凋亡损伤的作用机制尚不明确。
目的：探讨氟暴露对原代神经细胞线粒体相关内质网膜内Ca2+转运通道蛋白及细胞凋亡水平的影响。 
方法：体外培养新生SD大鼠原代神经细胞，培养至第7天采用神经元核特异性抗体NeuN行免疫荧光染色鉴定。神经细胞按照加氟浓度分为对照组(含0 mmol/L 氟化钠)、低氟组(含0.5 mmol/L 氟化钠)、高氟组(含1 mmol/L氟化钠)，氟暴露24 h后光镜下观察细胞形态变化；蛋白免疫印迹检测凋亡相关蛋白BAX/BCL-2和钙转移相关通路VDAC1、GRP75、IP3R蛋白表达；RT-PCR检测钙转移相关通路VDAC1、GRP75、IP3R mRNA表达；线粒体Ca2+探针Rhod-2AM检测Ca2+水平；线粒体膜电位试剂盒检测线粒体膜电位变化；流式细胞术、TUNEL染色法检测细胞凋亡水平。 
结果与结论：①培养至第7天的神经细胞经鉴定纯度可达90%以上，杂质少，生长状态良好，细胞网络连接紧密，达到后续实验要求；②与对照组相比，低氟组和高氟组神经细胞聚团生长增多，突起断裂，胞体圆缩，细胞之间连接网络被破坏；与低氟组相比，高氟组细胞损伤变化更为明显；③与对照组比较，低氟组和高氟组VDAC1、GRP75、IP3R蛋白表达上调(P < 0.05)，凋亡相关蛋白BAX/BCL-2比值升高(P < 0.05)；与对照组相比，低氟组VDAC1、GRP75 mRNA表达明显上调(P < 0.05)，高氟组VDAC1、GRP75、IP3R mRNA表达显著上调(P < 0.01)；④氟暴露处理后细胞凋亡水平明显上升，且高氟组显著高于对照组和低氟组(P < 0.01)；⑤氟暴露处理后神经细胞线粒体Ca2+浓度明显增加(P < 0.05)，线粒体膜电位下降(P < 0.01)，且高氟组损伤程度更为明显(P < 0.05)。结果表明，氟暴露会损害原代神经细胞的形态结构，导致细胞内质网-线粒体间Ca2+转移通路蛋白表达上调，线粒体Ca2+超载，线粒体损伤，细胞凋亡水平升高。

关键词: 氟暴露, 原代神经细胞, Ca2+转移, 细胞凋亡, 线粒体膜电位, 线粒体损伤, 线粒体相关内质网膜, 钙转运通道

Abstract: BACKGROUND: Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+ overload, but the mechanism of Ca2+ flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.
OBJECTIVE: To investigate the effect of fluoride exposure on Ca2+ transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells. 
METHODS: Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7. The nerve cells were divided into control group (containing 0 mmol/L sodium fluoride), low fluoride group (containing 0.5 mmol/L sodium fluoride), and high fluoride group (containing 1 mmol/L sodium fluoride). The cell morphological changes were observed by light microscope 24 hours after fluorine exposure. The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1, GRP 75, and IP3R were detected using western blot assay. The expression levels of VDAC1, GRP 75, and IP3R mRNA were detected by RT-PCR. Ca2+ levels were detected by Rhood-2AM Ca2+ probe. Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential. The level of apoptosis was determined by flow cytometry and TUNEL staining.  
RESULTS AND CONCLUSION: (1) The purity of neurons cultured on day 7 had been determined to be over 90%, with few impurities, good growth status, and tight cell network connections, meeting the requirements of subsequent experiments. (2) Compared with the control group, growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased; the processes were broken; the cell body was rounded, and the connection network between cells was destroyed. Compared with the low-fluoride group, the cell damage changes in the high-fluoride group were more obvious. (3) Compared with the control group, the protein expressions of VDAC1, GRP75, and IP3R were increased in the low-fluoride group and the high-fluoride group (P < 0.05), and the ratio of apoptosis-related protein BAX/BCL-2 was increased (P < 0.05). Compared with the control group, the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased (P < 0.05); the expression levels of VDAC1, GRP75, and IP3R mRNA in the high-fluoride group were significantly increased (P < 0.01). (4) The level of cell apoptosis increased significantly after fluoride exposure, and the high-fluoride group was significantly higher than the control and low-fluoride groups (P < 0.01). (5) After fluoride exposure, the concentration of mitochondrial Ca2+ in nerve cells increased significantly (P < 0.05), the mitochondrial membrane potential decreased (P < 0.01), and the degree of damage in the high-fluoride group was more obvious (P < 0.05). The results show that fluoride exposure impairs the morphological structure of primary neural cells, resulting in upregulation of Ca2+ transfer pathway protein expression between the endoplasmic reticulum and mitochondria, mitochondrial Ca2+ overload, mitochondrial damage, and increased levels of apoptosis.


Key words: fluoride exposure, primary nerve cell, Ca2+ transfer, apoptosis, mitochondrial membrane potential, mitochondrial damage, mitochondria-associated endoplasmic reticulum membrane, calcium transport channel

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