吉林大学学报(医学版) ›› 2024, Vol. 50 ›› Issue (1): 33-41.doi: 10.13481/j.1671-587X.20240105

• 基础研究 • 上一篇    下一篇

APOE多态性在炎症因子诱导的神经毒性反应性星形胶质细胞中的差异作用

王岩,李晓慧,季瑶,崔理立,蔡玉洁()   

  1. 广东医科大学附属医院 广东省衰老相关心脑疾病重点实验室,广东 湛江 524001
  • 收稿日期:2023-03-17 出版日期:2024-01-28 发布日期:2024-01-31
  • 通讯作者: 蔡玉洁 E-mail:caiyujie@gdmu.edu.cn
  • 作者简介:王 岩(1984-),女,甘肃省兰州市人,助理研究员,理学博士,主要从事阿尔茨海默病发病机制方面的研究。
  • 基金资助:
    国家自然科学基金项目(81671181);广东省科技厅自然科学基金项目(2022A1515010593);广东医学院科 研基金项目(GDMUM2020011)

Differential effects of APOE polymorphism in neurotoxicity-responsive astrocytes induced by inflammatory factor

Yan WANG,Xiaohui LI,Yao JI,Lili CUI,Yujie CAI()   

  1. Guangdong Provincial Key Laboratory of Age-Related Cardiac and Cerebral Diseases,Affiliated Hospital,Guangdong Medical University,Zhanjiang 524001,China
  • Received:2023-03-17 Online:2024-01-28 Published:2024-01-31
  • Contact: Yujie CAI E-mail:caiyujie@gdmu.edu.cn

摘要:

目的 探讨载脂蛋白E(APOE)基因多态性在神经毒性反应性星形胶质细胞中的差异作用,为阿尔茨海默病(AD)发病机制的研究提供理论依据。 方法 体外分离培养APOE基因敲除小鼠(APOE -/- )原代皮层星形胶质细胞,免疫荧光染色法鉴定细胞纯度。构建人APOE3和APOE4重组过表达质粒,分别转染至原代APOE -/- 星形胶质细胞中,以APOE -/- 原代细胞为对照,采用Western blotting法检测细胞中APOE和神经胶质酸性蛋白(GFAP)蛋白表达水平,采用酶联免疫吸附试验(ELISA)法检测细胞培养上清中APOE水平。采用白细胞介素1α(IL-1α)、肿瘤坏死因子(TNF)和补体C1q联合刺激转染APOE3和转染APOE4的原代星形胶质细胞制备炎症模型,分为APOE3+PBS组、APOE4+PBS组、APOE3+IL-1α+TNF+CC1q组和APOE4+IL-1α+ TNF+C1q组,采用细胞免疫荧光染色法观察各组细胞形态表现,采用实时荧光定量PCR(RT-qPCR)法检测各组细胞中磷脂酰肌醇蛋白聚糖4(Gpc4)、磷脂酰肌醇蛋白聚糖6(Gpc6)、血小板反应蛋白1(Thbs1)、血小板反应蛋白 2(Thbs2)、酸性分泌蛋白类似蛋白1(Sparcl1)、胶质细胞源性神经营养因子(GDNF)、C3和 S100钙结合蛋白B(S100B)mRNA表达水平,微球吞噬实验检测各组细胞吞噬能力,Western blotting法检测各组细胞中B细胞淋巴瘤2(Bcl-2)和含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)蛋白表达水平。 结果 与APOE -/- 组比较转染APOE3和APOE4组细胞中APOE和GFAP蛋白表达水平及细胞培养上清中APOE水平明显升高(P<0.01)。荧光显微镜下观察,分别与APOE3+PBS组和APOE4+PBS组比较,APOE3+IL-1α+TNF+Cq1组和APOE4+IL-1α+TNF+Cq1组星形胶质细胞突起变短,胞体变大;与APOE3+IL-1α+TNF+Cq1组比较,APOE4+IL-1α+TNF+Cq1组星形胶质细胞突起更短。分别与APOE3+PBS组和APOE4+PBS组比较,APOE3+IL-1α+TNF+Cq1组和APOE4+IL-1α+TNF+Cq1组细胞中Gpc4、Gpc6、Thbs1、Thbs2和Sparcl1 mRNA表达水平明显降低(P<0.01);与APOE3+IL-1α+TNF+Cq1组比较,APOE4+IL-1α+TNF+Cq1组细胞中Gpc4、Gpc6、Thbs1、Thbs2和Sparcl1 mRNA表达水平明显降低(P<0.05或P<0.01)。分别与APOE3+PBS组和APOE4+PBS组比较,APOE3+IL-1α+TNF+Cq1组和APOE4+IL-1α+TNF+Cq1组细胞中GDNF mRNA表达水平明显降低(P<0.01),C3和S100B mRNA表达水平明显升高(P<0.01);与APOE3+IL-1α+TNF+Cq1组比较,APOE4+IL-1α+TNF+Cq1组细胞中GDNF mRNA表达水平明显降低(P<0.05),C3和S100B mRNA表达水平明显升高(P<0.05)。分别与APOE3+PBS组和APOE4+PBS组比较,APOE3+IL-1α+TNF+Cq1组和APOE4+IL-1α+TNF+Cq1组细胞吞噬微珠数量明显减少;与APOE3+IL-1α+TNF+Cq1组比较,APOE4+IL-1α+TNF+Cq1组细胞吞噬微珠数量明显减少。分别与APOE3+PBS组和APOE4+PBS组比较,APOE3+IL-1α+TNF+Cq1组和APOE4+IL-1α+ TNF+Cq1组细胞中Bcl-2蛋白表达水平明显降低(P<0.05或P<0.01),Caspase-3蛋白表达水平明显升高(P<0.01);与APOE3+IL-1α+TNF+Cq1组比较,APOE4+IL-1α+TNF+Cq1组细胞中Bcl-2蛋白表达水平明显降低(P<0.01),Caspase-3蛋白表达水平明显升高(P<0.05)。 结论 APOE4基因型比APOE3诱导炎症因子能力更强,可诱导神经毒性反应性星形胶质细胞表型,增加神经毒性,影响星形胶质细胞凋亡,从而加剧神经元损伤。

关键词: 阿尔茨海默病, 星形胶质细胞, 载脂蛋白E, 基因多态性, 细胞凋亡, 神经毒性

Abstract:

Objective To discuss the differential effects of apolipoprotein E (APOE) gene polymorphism in the neurotoxicity-reactive astrocytes, and to provide the theoretical basis for the study of the pathogenesis of Alzheimer’s disease (AD). Methods The primary cortical astrocytes from the APOE-knockout mice (APOE -/- ) were isolated and cultured in vitro,and the purity of the cells was identified by immunofluorescence staining. The human APOE3 and APOE4 recombinant over-expression plasmids were constructed and separately transfected into the primary APOE -/- astrocytes, and the APOE -/- primary cells were regarded as control. Western blotting method was used to detect the expression levels of APOE and glial fibrillary acidic protein (GFAP) proteins in the cells; enzyme-linked immunosorbent assay (ELISA) method was used to detect the APOE level in the cellular culture supernatant. The inflammatory models were prepared with the primary astrocytes transfected with APOE3 and APOE4 and co-stimulated with interleukin-1α(IL-1α), tumor necrosis factor (TNF), and complement C1q.The cells were divided into APOE3+PBS group, APOE4+PBS group, APOE3+IL-1α+TNF+C1q group, and APOE4+IL-1α+TNF+C1q group. Cell immunofluorescence staining method was used to observe the morphology of the cells in various groups; real-time fluorescence quantitative PCR (RT-qPCR) method was used to detect the expression levels of glypican 4 (Gpc4), glypican 6 (Gpc6), thrombospondin 1 (Thbs1), thrombospondin 2 (Thbs2), SPARC-like protein 1 (Sparcl1) and glial cell line derived neurotrophic factor (GDNF), C3,and S100 calcium binding protein B (S100B) mRNA in the cells in various groups; microsphere phagocytosis assay was used to detect the phagocytic capacities of the cells in various groups; Western blotting was used to detect the protein expression levels of B-cell lymphoma 2 (Bcl-2),and cysteinyl aspartate specific protease-3 (Caspase-3) proteins in the cells in various groups. Results Compared with APOE -/- group, the expression levels of APOE and GFAP proteins in the cells and the APOE level in the cellular culture supernatant in transfected APOE3 and transfected APOE4 groups were increased (P<0.01). The fluorescence microscope observation results showed that compared with APOE3+PBS and APOE4+PBS groups, the astrocytic processes in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group became shorter and the cell bodies became larger; compared with APOE3+IL-1α+TNF+Cq1 group, the astrocytic processes in APOE4+IL-1α+TNF+Cq1 group were even shorter. Compared with APOE3+PBS and APOE4+PBS groups, the expression levels of Gpc4, Gpc6, Thbs1, Thbs2,and Sparcl1 mRNA in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group were significantly decreased (P<0.01); compared with APOE3+IL-1α+TNF+Cq1 group, the expression levels of Gpc4, Gpc6,Thbs1, Thbs2, and Sparcl1 mRNA in the cells in APOE4+IL-1α+TNF+Cq1 group were significantly decreased (P<0.05 or P<0.01). Compared with APOE3+PBS and APOE4+PBS groups, the expression levels of GDNF mRNA in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group were decreased(P<0.01),and the expression levels of C3 and S100B mRNA were increased(P<0.01);compared with APOE3+IL-1α+TNF+Cq1 group, the expression level of GDNF mRNA in the cells in APOE4+IL-1α+TNF+Cq1 group was decreased(P<0.05),and the expression levels of C3 and S100B mRNA were increased(P<0.05). Compared with APOE3+PBS group and APOE4+PBS group,the numbers of hagocytosis of microspheres in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group were significantly decreased; compared with APOE3+IL-1α+TNF+Cq1 group,the number of hagocytosis of microspheres in the cells in APOE4+IL-1α+TNF+Cq1 group was significantly decreased. Compared with APOE3+PBS group and APOE4+PBS group,the expression levels of Bcl-2 protein in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group were decreased (P<0.05 or P<0.01)and the expression levels of Caspase-3 protein were significantly increased(P<0.01);compared with APOE3+IL-1α+TNF+Cq1 group, the expression level of Bcl-2 protein in the cells in APOE4+IL-1α+TNF+Cq1 group was decreased(P<0.01),and the expression level of Caspase-3 protein was increased(P<0.05). Conclusion The APOE4 genotype has a stronger ability to induce the inflammatory factors compared with APOE3;it can lead to a neurotoxicity-reactive astrocyte phenotype,increase the neurotoxicity,affect the astrocyte apoptosis, and aggravate the neuron damage.

Key words: Alzheimer’s disease, Astrocyte, Apolipoprotein E, Gene polymorphism, Apoptosis, Neurotoxicity

中图分类号: 

  • R742