解剖学报

解剖学报 ›› 2021, Vol. 52 ›› Issue (2): 161-167.doi: 10.16098/j.issn.0529-1356.2021.02.001

• 神经生物学 •    下一篇

铁转运相关蛋白在肌萎缩性侧索硬化症转基因鼠脊髓中的表达变化

张雅雯1,5 高莹2,5 孙菡聪3,5 张皓云4,5* 王凤斌1*    

  1. 1.潍坊医学院基础医学院生理学教研室; 2.生命科学与技术学院2018级生物医学工程; 3.临床医学院2018级临床医学; 4.基础医学院基础医学概论教研室; 5.山东省神经疾病与再生修复重点实验室，山东 潍坊 261053
  • 收稿日期:2020-07-02 修回日期:2020-09-08 出版日期:2021-04-06 发布日期:2021-04-06
  • 通讯作者: 张皓云;王凤斌 E-mail:haoyunzh@wfmc.edu.cn
  • 基金资助:
    国家自然科学基金;潍坊医学院博士启动基金

Expressions of iron transport related proteins in the spinal cord of amyotrophic lateral sclerosis transgenic mice

ZHANG Ya-wen1,5 GAO Ying2,5 SUN Han-cong3,5 ZHANG Hao-yun4,5* WANG Feng-bin1*   

  1. 1.Division of Physiology, School of Basic Medical Sciences;2.Biomedical Engineering, Grade 2018, School of Life Science and Technology; 3.Clinical Medicine, Grade 2018, School of Clinical Medicine;  4.Division of Preclinical Medicine Theories, School of Basic Medical Sciences;5.Shandong Province Key Laboratory of Neurologic Disorders and Regenerative Repair, Weifang Medical University, Shangdong Weifang 261053, China
  • Received:2020-07-02 Revised:2020-09-08 Online:2021-04-06 Published:2021-04-06
  • Contact: Hao-yun;WANG Feng-bin E-mail:haoyunzh@wfmc.edu.cn

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摘要：

目的 探讨肌萎缩性侧索硬化症（ALS）转基因鼠脊髓内铁转运相关蛋白表达变化与铁稳态失衡的关联。 方法 选取hSOD1G93A转基因鼠（ALS鼠）和同窝野生型鼠（WT鼠），分别于生后70、95和122 d分离脊髓，每时间点每组各9只实验动物。Western blotting检测脊髓组织内铁转运蛋白二价金属转运蛋白-1（DMT1）、铁转运蛋白-1（FPN1）及调节蛋白铁调节蛋白-1（IRP1）的表达；免疫荧光双重标记检测脊髓腰段前角内细胞共定位情况。   结果 Western blotting显示，与WT鼠比较，各时间点ALS鼠脊髓内DMT1表达均显著降低（P<0.05，P<0.01）；70 d FPN1表达升高（P<0.05），95 d和122 d表达下降（P<0.01）；95 d、122 d IRP1表达降低（P<0.01）。免疫荧光双重标记显示，在70 d WT鼠和ALS鼠腰段脊髓中DMT1主要与β-微管蛋白Ⅲ（β-tubulin Ⅲ）共表达。与WT组相比，95 d ALS鼠脊髓腰段前角神经元内DMT1免疫反应强，而FPN1荧光强度减弱。随疾病进展，DMT1、FPN1与反应性胶质细胞共定位表达增多。IRP1随疾病进展表达强度降低。  结论 随ALS病程进展，发病早期神经元铁转入增加，转出减少，反应性神经胶质细胞铁转运活性增强，参与局部铁稳态失衡及脊髓前角运动神经元进行性丢失。IRP1表达降低，部分参与局部铁代谢调节。

关键词: 肌萎缩侧索硬化症, 铁稳态, 二价金属转运蛋白-1, 铁转运蛋白1, 铁调节蛋白, 免疫印迹法, 小鼠

Abstract:

Objective  To investigate the relationship between the expressions of iron transport related proteins and the dysregulation of iron homeostasis in the spinal cord of amyotrophic lateral sclerosis(ALS) transgenic mice.  Methods The hSOD1G93A transgenic mice (ALS mice) and littermate wild-type mice (WT mice) were selected to separate the spinal cord at day 70, day 95, and day 122 after birth, 9 mice per time point and per group. Western blotting was used to detect the expressions of iron transporter divalent metal transporter-1 (DMT1), ferroportin 1 (FPN1) and regulatory protein iron regulatory protein 1(IRP1) in the spinal cord. Double immunofluorescence labeling was used to detect the co-localization of cells in the ventral horn of lumbar spinal cord.    Results Western blotting results  showed that compared with WT mice the expressions of DMT1 protein were down-regulated with the disease progression from day 70 to day 122 (P<0.05, P<0.01); FPN1 protein was transiently up-regulated at day 70 (P<0.05), and decline expressions were observed at day 95 and day 122 (P<0.01); IRP1 was down-regulated at day 95 and day 122 (P<0.01). Double immunofluorescence labeling revealed that at day 70, DMT1 co-expressed mainly with β-tubulin Ⅲ both in WT and ALS mice lumbar spinal cord. Compared with the WT group, the DMT1 immunoreactivity in the neurons of the ventral horn lumbar spinal cord of ALS mice was elevated at day 95, while the FPN1 fluorescence intensity was weak. With the disease progression, the co-localization expression of DMT1, FPN1 with reactive glial cells increased. With the disease progresses, the expression of IRP1 decreased.    Conclusion With the progression of ALS, iron influx increases and iron outflux decreases in neurons at the early-symptomatic stage of ALS, the activity of iron transport in reactive glial cells is enhanced, which participates in local iron homeostasis imbalance and progressive loss of motor neurons in ventral horn of spinal cord. Decreased expression of IRP1 partly participates in the regulation of local iron metabolism.

Key words: Amyotrophic lateral sclerosis, Iron homeostasis, Divalent metal transporter-1, Ferroportin 1, Iron regulatory protein, Western blotting, Mouse

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