C3H1型锌指结合蛋白36介导的星形胶质细胞活化在肌萎缩侧索硬化症运动神经元退变中的作用

doi:10.16098/j.issn.0529-1356.2022.03.001

解剖学报 ›› 2022, Vol. 53 ›› Issue (3): 273-280.doi: 10.16098/j.issn.0529-1356.2022.03.001

• 神经生物学 • 下一篇

C3H1型锌指结合蛋白36介导的星形胶质细胞活化在肌萎缩侧索硬化症运动神经元退变中的作用

丁康^1,4张烽萍^1,4 齐高秀^1,2 林盟^1,4 陈敏^1,2 陈燕春^3,4 郭章玉^1,4 周风华^1,4* 管英俊^3,4*

1.潍坊医学院基础医学院临床病理系; 2.潍坊医学院附属医院病理科; 3.潍坊医学院基础医学院组织学与胚胎学教研室; 4.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:2021-08-20 修回日期:2021-10-10 出版日期:2022-06-06 发布日期:2019-06-06
通讯作者: 周风华;管英俊 E-mail:zhoufh@wfmc.edu.cn
基金资助:
国家自然科学基金;山东省自然科学基金;山东省自然科学基金;山东省自然科学基金;山东省高等学校青创科技支持计划;山东省高校科技发展项目重点项目;山东省医药卫生科技发展计划项目

Role of zinc finger protein 36, C3H type-like 1 mediating astrocytes activation in motor neuron degeneration in amyotrophic lateral sclerosis

DING Kang^1,4 ZHANG Feng-ping^1,4 QI Gao-xiu^1,2 LIN Meng^1,4 CHEN Min^1,2 CHEN Yan-chun^3,4 GUO Zhang-yu^1,4 ZHOU Feng-hua^1,4* GUAN Ying-jun^3,4*

1.Department of Clinical Pathology, School of Basic Medicine, Weifang Medical University; 2.Department of Pathology, Affiliated Hospital of Weifang Medical University; 3.Department of Histology and Embryology, School of Basic Medicine, Weifang Medical University; 4.Neurologic Disorders and Regenerative Repair Key Laboratory of Shandong Province，Shandong Weifang 261053,China

Received:2021-08-20 Revised:2021-10-10 Online:2022-06-06 Published:2019-06-06
Contact: ZHOU Feng-hua;GUAN Ying-jun E-mail:zhoufh@wfmc.edu.cn

摘要/Abstract

摘要：

目的探讨C3H1型锌指结合蛋白36（ZFP36L1）介导的星形胶质细胞活化在肌萎缩侧索硬化症（ALS）运动神经元退变中的作用。方法以铜锌超氧化物歧化酶1（SOD1)-G93A转基因小鼠作为动物模型，同窝野生型小鼠作为对照（突变型及野生型小鼠各时间点分别取13只小鼠）；Real-time PCR、Western blotting检测小鼠发病早期、中期及晚期脊髓内ZFP36L1 mRNA及蛋白变化，免疫荧光染色检测ZFP36L1在脊髓内的表达及分布；出生后 1～2 d新生小鼠15只，建立SOD1-G93A突变型原代星形胶质细胞模型，Real-time PCR、Western blotting检测星形胶质细胞内ZFP36L1 mRNA及蛋白水平的变化，si-ZFP36L1转染SOD1-G93A突变型原代星形胶质细胞，Western blotting检测转染效率，Western blotting及ELISA检测转染后星形胶质细胞分泌炎性因子肿瘤坏死因子α（TNF-α）、白细胞介素18（IL-18）变化；沉默SOD1-G93A突变型原代星形胶质细胞内ZFP36L1后，与SOD1-G93A突变型NSC34细胞共培养，通过5’-乙炔基-2’-脱氧尿苷（EdU）实验和观察增殖细胞核抗原（PCNA）的水平明确ZFP36L1对NSC34细胞增殖的影响，通过TUNEL实验及观察剪切Caspase-3（cleaved-Caspase-3）的水平明确ZFP36L1对NSC34细胞凋亡的影响，以转染空白小干扰RNA（siRNA）作为对照组。结果与野生型小鼠相比，ZFP36L1在SOD1-G93A转基因小鼠脊髓组织内mRNA及蛋白水平均下调，在野生型小鼠脊髓组织内，ZFP36L1主要与β-微管蛋白Ⅲ（β-tubulin Ⅲ）阳性的神经元共表达，而SOD1-G93A突变型小鼠的脊髓组织内，ZFP36L1在神经元内表达减弱，与胶质纤维酸性蛋白（GFAP）标记的星形胶质细胞共表达明显增加；SOD1-G93A突变型原代星形胶质细胞内ZFP36L1表达增加，si-ZFP36L1能明显降低SOD1-G93A突变型原代星形胶质细胞内ZFP36L1水平；沉默ZFP36L1后星形胶质细胞分泌炎性因子 TNF-α、 IL-18明显降低。此外，沉默ZFP36L1后，SOD1-G93A突变型原代星形胶质细胞能显著增强NSC34细胞增殖活性，抑制NSC34细胞凋亡。结论在ALS发病过程中，星形胶质细胞被激活，ZFP36L1通过星形胶质细胞分泌的炎性因子促进了ALS运动神经元的退变。

关键词: 肌萎缩侧索硬化症, C3H1型锌指结合蛋白36, 炎性因子, 运动神经元, 星形胶质细胞, 免疫荧光, 转基因小鼠

Abstract:

Objective To investigate the role of zinc finger protein 36,C3H type-like 1（ZFP36L1）mediating astrocytes activation in the degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Methods Superoxide dismutase 1 (SOD1)-G93A transgenic mice were used as animal models, the wild-type littermates as the control (13 mice were taken from mutant and wild-type mice at each time point). The ZFP36L1 mRNA and protein levels of the spinal cord in the early, middle and late stage were detected by Real-time PCR and Western blotting. The expression and distribution of ZFP36L1 in the spinal cord were detected by immunofluorescence. Primary astrocyte model was established from 15 postnatal 1-2 day mice. The ZFP36L1 mRNA and protein levels in astrocytes were detected by Real-time PCR and Western blotting. Si-ZFP36L1 was transfected into SOD1-G93A mutant primary astrocytes. The transfection efficiency was detected by Western blotting. Tumor necrosis factor α (TNF-α) and interleukin-18 (IL-18) secreted from astrocytes after transfection were assessed by Western blotting and ELISA. After silencing ZFP36L1 in SOD1-G93A mutant primary astrocytes, it was cocultured with SOD1-G93A mutant NSC34 cells. 5’-ethyyl-2’deoxyuridine (EdU) test and the level of proliferating cell nuclear antigen (PCNA) were used to determine the effect of ZFP36L1 on NSC34 cell proliferation. TUNEL test and the level of cleaved-Caspase-3 were used to determine the effect of ZFP36L1 on NSC34 cell apoptosis. Blank small interfering RNA(siRNA) was transfected as the control group. Results Compared with the wild-type mice, the mRNA and protein levels of ZFP36L1 were downregulated in the spinal cord of SOD1-G93A transgenic mice. In wild type mice, ZFP36L1 positive cells were mainly β-tubulin Ⅲ positive. In SOD1-G93A mutant mice, ZFP36L1 positive cells were mainly glial fibrillary acidic protein (GFAP) positive. The expression of ZFP36L1 in SOD1-G93A mutant primary astrocytes increased, and si-ZFP36L1 reduced the level of ZFP36L1 in SOD1-G93A mutant primary astrocytes significantly. Inflammatory factors including TNF-α, IL-18 decreased significantly after silencing ZFP36L1. In addition, after silencing ZFP36L1 expression, SOD1-G93A mutant primary astrocytes enhanced the proliferation activity of NSC34 cells and inhibited NSC34 cell apoptosis significantly. Conclusion Astrocytes are activated in the process of ALS. ZFP36L1 promotes the degeneration of motor neurons in ALS through the inflammatory factors secreted by astrocytes.

Key words: Amyotrophic lateral sclerosis, Zinc finger protein 36, C3H type-like 1, Inflammatory factor, Motor neuron, Astrocyte, Immunofluorescence, Transgenic mouse

中图分类号:

R744.8

丁康张烽萍齐高秀林盟陈敏陈燕春郭章玉周风华管英俊. C3H1型锌指结合蛋白36介导的星形胶质细胞活化在肌萎缩侧索硬化症运动神经元退变中的作用[J]. 解剖学报, 2022, 53(3): 273-280.

DING Kang ZHANG Feng-ping QI Gao-xiu LIN Meng CHEN Min CHEN Yan-chun GUO Zhang-yu ZHOU Feng-hua GUAN Ying-jun. Role of zinc finger protein 36, C3H type-like 1 mediating astrocytes activation in motor neuron degeneration in amyotrophic lateral sclerosis[J]. Acta Anatomica Sinica, 2022, 53(3): 273-280.

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C3H1型锌指结合蛋白36介导的星形胶质细胞活化在肌萎缩侧索硬化症运动神经元退变中的作用

Role of zinc finger protein 36, C3H type-like 1 mediating astrocytes activation in motor neuron degeneration in amyotrophic lateral sclerosis

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