脑型疟炎症微环境诱导星形胶质细胞活化及对神经元损伤的研究  

Activation of astrocytes and neuronal damage induced by the inflammatory microenvironment of cerebral malaria

作　　者：仝国栋 朱卿昊 王军 刘晓冉 沈燕 梁姣 李英辉 黄豫晓 王一 赵亚 TONG Guodong;ZHU Qinghao;WANG Jun;LIU Xiaoran;SHEN Yan;LIANG Jiao;LI Yinghui;HUANG Yuxiao;WANG Yi;ZHAO Ya(The College of Life Sciences,Northwest University,Xi’an 710127,Shaanxi,China;Department of Medical Microbiology and Parasitology,Basic Medical College,Air Force Medical University,Xi’an 710032,Shaanxi,China;Grade 2019 Clinical Medicine(Five-Year Program),Basic Medical College,Air Force Medical University,Xi’an 710032,Shaanxi,China)

机构地区：[1]西北大学生命科学学院,陕西西安710127 [2]空军军医大学基础医学院微生物与病原生物学教研室,陕西西安710032 [3]空军军医大学基础医学院,陕西西安710032

出　　处：《中国寄生虫学与寄生虫病杂志》2024年第2期160-168,共9页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(82002158,82072298);陕西省自然科学基础研究计划-重点项目(2022JZ-14)。

摘　　要：目的探索脑型疟发病过程中,星形胶质细胞被脑血管周炎性微环境诱导活化及其机制,及其对神经元损伤的影响。方法4~5周C57BL/6雄性小鼠经腹腔接种5×10^(6)个感染伯氏疟原虫ANKA株的红细胞,7~10 d后死亡,作为实验型脑型疟模型。新生3~5 d的乳鼠,安乐死后分离脑皮层原代星形胶质细胞;24 h内的乳鼠,安乐死后分离脑皮层原代神经元。以20 ng/mlγ干扰素(IFN-γ)、1 ng/ml肿瘤坏死因子α(TNF-α)和感染疟原虫的小鼠红细胞(pRBC)诱导星形胶质细胞活化,作为活化组,同时设置未处理静息态细胞作为对照组。24 h后用细胞裂解提取液提取总RNA测序,聚类分析生成热图,选取代表性的数个基因绘制小提琴图。ELISA测定细胞培养上清中CXCL10表达水平。分离脑型疟小鼠脾脏CD8^(+)T细胞,与活化的星形胶质细胞共孵育,荧光显微镜下观察免疫突触及与CXCL10抗体共孵育后的CXCL10水平,流式细胞仪检测CD80等分子表达水平。原代神经元加入两组星形胶质细胞培养上清,在MAP2抗体中孵育,设置空白培养基作为空白组,乳酸脱氢酶(LDH)检测试剂盒检测神经元损伤程度,CCK-8试剂盒检测神经元活力。Western blotting检测STAT1、STAT3及其磷酸化分子水平,活化组加入STAT1通路抑制剂氟达拉滨,免疫荧光染色观察神经元形态。采用PRISM Graph Pad 8.0软件进行统计学分析,两两比较采用t检验。结果活化组星形胶质细胞形态由扁平星状变为长梭形,胶质纤维酸性蛋白(GFAP)相对含量为(1.36±0.03),高于对照组的(1.00±0.00)(t=13.33,P<0.01);活化组细胞培养上清中的CXCL10含量为(7.07±0.81)ng/ml,高于对照组的(2.57±0.28)ng/ml(t=9.05,P<0.01)。转录组分析显示,活化组抗原加工、提呈与T细胞趋化因子转录水平上调(均P<0.01),CD80、CD86、MHCⅠ表达水平上调。活化组和CD8^(+)T细胞共孵育形成“免疫突触”,CD8^(+)T细胞CD69表达量提高。LDH结果显示,活化组上Objective To explore the activation of astrocytes induced by the perivascular inflammatory microenvironment during the pathogenesis of the brain during the pathogenesis process of cerebral malaria and its mechanism,as well as its impact on neuronal damage.Methods C57BL/6 male mice aged 4-5 weeks were intraperitoneally inoculated with 5×10^(6)red blood cells infected with the ANKA strain of Plasmodium berghei,which would be died 7-10 d later and served as an experimental cerebral malaria model.Newborn suckling mice aged 3-5 d were euthanized to isolate primary astrocytes from the cerebral cortex;while the other suckling mince were euthanized within 24 hours for isolation of primary cortical neurons.The cell culture containing 20 ng/mlγinterferon(IFN-γ),1 ng/ml tumor necrosis factorα(TNF-α)and mice red blood cells(pRBC)infected with malaria parasites were used to induce activation of astrocytes,and assigned as the activation group,while untreated resting-state cells were set as the control group.After cultivation for 24 h,total RNA was extracted by cell lysis and sequenced.Cluster analysis was performed to generate heatmaps,and representative genes were selected to draw violin diagram.ELISA was used to determine the expression level of CXCL10 in cell culture supernatant.The spleen CD8^(+)T cells of mice with cerebral malaria were separated and co-cultured with activated astrocytes to observe immune synapses and CXCL10 levels after co-incubation with CXCL10 antibodies by fluorescence microscopy,and CD80 and other molecular expression levels were detected using flow cytometry.Primary neurons were added into two sets of astrocyte culture supernatants and incubated in MAP2 antibody.Blank culture medium was set as the blank group,and lactate dehydrogenase(LDH)and CCK-8 assay kit were used to detect the degree of neuronal damage.Western blotting was used to detect the levels of STAT1,STAT3,and their phosphorylated molecules.The activation group was treated with STAT1 pathway inhibitor fludarabine,and the morphology

关 键 词：脑型疟 星形胶质细胞 CD8^(+)T细胞 神经元 炎性微环境 

分 类 号：R531.33[医药卫生—内科学]