基于核糖核酸测序探讨马尔尼...感染致小鼠脑皮层损伤的机制_韦雪芹.pdf

论著基础研究基金项目:国家自然科学基金(31970167);广西自然科学基金(2022GXNSFBA035660)第一作者简介:韦雪芹，在读硕士研究生，研究方向:传染病流行病学。通信作者简介:叶力，博士，教授，研究方向:传染病流行病学、病毒免疫学和分子病毒学。基于核糖核酸测序探讨马尔尼菲篮状菌感染致小鼠脑皮层损伤的机制韦雪芹1袁宗祥1，2张君涵1李炫蓉1韦吴迪1，2，3蒋俊俊1，2，3王凤仪1莫初叶1康旖雯1梁浩1，2，3叶力1，2，3 1 广西医科大学公共卫生学院广西艾滋病防治研究重点实验室，广西南宁市530021;2 中国(广西)-东盟新发传染病联合实验室，广西南宁市530021;3 广西医科大学再生医学与医用生物资源开发应用省部共建协同创新中心，广西南宁市530021【摘要】目的应用核糖核酸测序(NA-seq)技术探讨马尔尼菲篮状菌(TM)致小鼠脑皮层损伤的可能机制。方法(1)将 10 只 C57/BL6 小鼠随机分为 TM 感染组和健康对照组(5 只/组)后，分别经尾静脉注射TM 分生孢子悬液、生理盐水。将 BV2 细胞和 N2a 细胞均随机分为 TM 感染组和空白对照组，TM 感染组加入TM 分生孢子悬液，空白对照组加入等量培养液。(2)建模成功后，取小鼠脑皮层组织进行 NA-seq 后分析差异表达基因(DEGs);采用 4 1 2 软件对 DEGs 进行京都基因与基因组百科全书(KEGG)通路富集分析和基因本体论(GO)功能富集分析等。取两组小鼠脑皮层组织及两组 BV2 细胞、N2a 细胞，采用实时荧光定量 PC对富集通路相关 DEGs、促炎因子进行体内体外验证。结果(1)共获得871 个 DEGs，其中上调基因373 个、下调基因 498 个。KEGG 通路富集分析结果显示，显著性最大的通路为氧化磷酸化通路(Q=9 60 103)，基因数目比例最大的通路为神经退行性病变-多种疾病通路;GO 功能富集分析结果显示，DEGs 主要涉及细胞因子产生的正向调节、突触组织等生物学过程和组织。(2)体内体外验证结果表明，与对应对照组相比，TM 感染组小鼠脑皮层组织和 N2a 细胞中 Atp4a、Abcg3 的 mNA 表达水平均上调，Med8 mNA 表达水平下调，N2a 细胞中 Ifi47、AA388235 的 mNA 表达水平上调(均 P 0 05)，TM 感染组小鼠脑皮层组织及 BV2 细胞中白细胞介素 6、白细胞介素 1 和肿瘤坏死因子 的 mNA 表达水平均上调(均 P 0 05)，均与 NA-seq 结果一致。结论TM感染可能通过直接改变神经元细胞糖代谢，或间接激活小胶质细胞炎症反应，从而导致脑皮层损伤。【关键词】马尔尼菲篮状菌;脑皮层损伤;核糖核酸测序;氧化磷酸化;炎症反应;小鼠;细胞实验【中图分类号】331;519【文献标识码】A【文章编号】0253-4304(2023)03-0289-07DOI:10 11675/j issn 0253-4304 2023 03 08Mechanism of cerebral cortex injury in mice induced by Talaromyces marneffei infection:an exploration based on NA sequencingWEI Xueqin1，YUAN Zongxiang1，2，ZHANG Junhan1，LI Xuanrong1，WEI Wudi1，2，3，JIANG Junjun1，2，3，WANG Fengyi1，MO Chuye1，KANG Yiwen1，LIANG Hao1，2，3，YE Li1，2，3(1 Guangxi Key Laboratory of AIDS Prevention and Control，School of Public Health，Guangxi Medical University，Nanning 530021，Guangxi，China;2 China Guangxi-ASEAN Joint Laboratory of Emerging Infectious Diseases，Nanning 530021，Guangxi，China;3 Collaborative Innovation Center of egenerative Medicine and Medical Bioresource Development and ApplicationCo-constructed by the Province and Ministry，Guangxi Medical University，Nanning 530021，Guangxi，China)【Abstract】ObjectiveTo explore the possible mechanism of cerebral cortex injury in mice induced byTalaromyces marneffei(TM)infection using NA sequencing(NA-seq)technique Methods(1)A total of10 C57/BL6 mice were randomly assigned to TM infection group or healthy control group，with 5 mice in each group，after that they received injection of TM conidial suspension or normal saline through the caudal vein，respectivelyBV2 cells and N2a cells were randomly assigned to TM infection group or blank control group，and TM conidialsuspension was added to the TM infection group，as well as culture solution with equivalent volume was added to theblank control group(2)After successful modeling，the differentially expressed genes(DEGs)were analyzed afterperforming NA-seq by obtaining cerebral cortex tissues in mice The Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and Gene Oncology(GO)functional enrichment analysis，etc on DEGs were performedby the 4 1 2 software The cerebral cortex tissues of mice in both groups，and BV2 and N2a cells in both groups982广西医学2023 年 2 月第 45 卷第 3 期were obtained，and enrichment pathway-related DEGs and proinflammatory cytokines were verified in vivo and in vitroby the real-time fluorescent quantitative PC esults(1)A total of 871 DEGs were obtained，therein，there were373 up-regulated genes and 498 down-regulated genes The results of KEGG pathway enrichment analysis revealed that themost significant pathway was oxidative phosphorylation pathway(Q=9 60 103)，and the pathway with the largestproportion of gene number was neurodegenerative-multiple diseases pathway The results of GO analysis interpreted thatDEGs were mainly involved in the biological processes and tissues in terms of positive regulation produced by cytokines，andsynaptic tissues，etc(2)The results of verification in vivo and in vitro implied that the TM infection group exhibitedup-regulated mNA expressions of Atp4a，Abcg3 in mice cerebral cortex tissues and in N2a cells，and a down-regulatedmNA expression of Med8 in mice cerebral cortex tissues and in N2a cells，as well as up-regulated mNA expressionsof Ifi47 and AA388235 in N2a cells as compared with the corresponding control groups(all P 0 05)The TMinfection group yielded up-regulated mNA expressions of interleukin 6，interleukin 1，and tumor necrosis factor in mice cerebral cortex tissues and in BV2 cells as compared with the corresponding control groups(all P 0 05)All results were consistent with NA-seq results ConclusionTM infection may directly change the glucose metabolismof neuron cells，or indirectly activate the microglial inflammatory response，so as to lead to cerebral cortex injury【Key words】Talaromyces marneffei，Cerebral cortex injury，NA sequencing，Oxidative phosphorylation，Inflammatory response，Mice，Cell experiment马尔尼菲篮状菌(Talaromyces marneffei，TM)，旧称马尔尼菲青霉菌，最早于 1956 年从野生越南中华竹鼠肝脏中分离出1，是一种温度双相型真菌。TM感染多发于 HIV 感染者或免疫力低下人群，感染病例主要分布在东南亚、印度北部及中国西南部等地区2。但近年来，在非 HIV 感染甚至免疫力功能正常的人群中 TM 感染病例的报告逐渐增多3 4。目前，研究证实 TM 感染可累及宿主多个组织和器