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-tubulinⅢ)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�
Objective To investigate the role of zinc finger protein 36,C3 H type-like 1(ZFP36 L1)mediating astrocytes activation in the degeneration of motor neurons in amyotrophic lateral sclerosis(ALS).Methods Superoxide dismutase 1(SOD1)-G93 A 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 ZFP36 L1 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 ZFP36 L1 in the spinal cord were detected by immunofluorescence.Primary astrocyte model was established from 15 postnatal 1-2 day mice.The ZFP36 L1 mRNA and protein levels in astrocytes were detected by Real-time PCR and Western blotting.Si-ZFP36 L1 was transfected into SOD1-G93 A mutant primary astrocytes.The transfection efficiency was detected by Western blotting.Tumor necrosis factorα(TNF-α)and interleukin-18(IL-18)secreted from astrocytes after transfection were assessed by Western blotting and ELISA.After silencing ZFP36 L1 in SOD1-G93 A mutant primary astrocytes,it was cocultured with SOD1-G93 A mutant NSC34 cells.5’-ethynyl-2’deoxyuridine(EdU)test and the level of proliferating cell nuclear antigen(PCNA)were used to determine the effect of ZFP36 L1 on NSC34 cell proliferation.TUNEL test and the level of cleaved-Caspase-3 were used to determine the effect of ZFP36 L1 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 ZFP36 L1 were downregulated in the spinal cord of SOD1-G93 A transgenic mice.In wild type mice,ZFP36 L1 positive cells were mainlyβ-tubulinⅢpositive.In SOD1-G93 A mutant mice,ZFP36 L1 positive cells were mainly glial fibrillary acidic protein(GFAP)positive.The expression of ZFP36 L1 in SOD1-G93 A mutant primary astrocytes increased,and si-ZFP36 L1 reduced the level of ZFP36 L1 in SOD1-G93 A mutant primary astrocyt
作者
丁康
张烽萍
齐高秀
林盟
陈敏
陈燕春
郭章玉
周风华
管英俊
DING Kang;ZHANG Feng-ping;QI Gao-xiu;LIN Meng;CHEN Min;CHEN Yan-chun;GUO Zhang-yu;ZHOU Feng-hua;GUAN Ying-jun(Department of Clinical Pathology,School of Basic Medicine,Weifang Medical University;Department of Pathology,Affiliated Hospital of Weifang Medical University;Department of Histology and Embryology,School of Basic Medicine,Weifang Medical University;Neurologic Disorders and Regenerative Repair Key Laboratory of Shandong Province,Shandong Weifang 261053,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2022年第3期273-280,共8页
Acta Anatomica Sinica
基金
国家自然科学基金(81871006)
山东省自然科学基金(ZR2020MH149,ZR2020MH150,ZR2019BH060)
山东省医药卫生科技发展计划项目(2019WS606)
山东省高校科技发展项目重点项目(J18KZ013)
山东省高等学校青创科技支持计划(2019KJK004)。
