【作者】 张锋；

【导师】 谭政；

【作者基本信息】 华中科技大学 ， 免疫学， 2019， 硕士

【摘要】 目的:ASIC1a（acid-sensitive ion channel 1a）是酸敏感离子通道家族成员,在外周和中枢神经系统中广泛表达,此外还发现在小鼠树突状细胞（DC）、巨噬细胞、肺动脉平滑肌细胞以及软骨细胞等均有表达,因其与多种疾病的发生与发展密切相关而具有重要生理学意义。巨噬细胞（macrophage）是天然免疫的重要组成部分,激活状态下分为M1型和M2型,在机体中对清除病原体、炎症及组织修复相关的初级免疫应答发挥重要作用。本课题组前期研究发现,巨噬细胞表达功能性ASIC1,ASIC1a敲除可上调M1型巨噬细胞膜分子CD80、CD86的表达,提示ASIC1a可能存在抑制M1型巨噬细胞的抗原提呈作用;ASIC1a在M2型巨噬细胞极化过程作用如何,并未有研究。本课题旨在研究ASIC1a在小鼠M2型骨髓来源巨噬细胞（bone marrow derived macrophage,BMM）和M2型腹腔巨噬细胞（peritoneal macrophage,PM）极化过程中是否发挥作用,并进一步探讨ASIC1a影响M2型极化过程的机制。研究方法:取野生型（WT,Wild Type）与ASIC1a^-/-小鼠诱导培养BMM与PM,流式细胞术检测F4-80标记阳性巨噬细胞进行鉴定。IL-4刺激24h分别诱导M2型BMM与PM,RT-PCR检测M2型巨噬细胞标记物Arg1、FIZZ1、YM1、Mgl1和Mgl2 mRNA表达水平,免疫印记法（WB）检测M2型巨噬细胞标记蛋白Arg1及巨噬细胞ASIC1蛋白表达,流式细胞术检测M2型巨噬细胞表面分子CD206与胞内分子FIZZ1表达。IL-4刺激BMM与PM不同时间点30min、60min、180min后,WB检测M2型巨噬细胞极化过程信号通路中STAT6磷酸化蛋白水平。实验所得数据用平均值±标准差表示,采用统计学软件GraphPad Prism 6进行双因素方差分析（Two way ANOVA）,两两比较采用student’s t检验,P﹤0.05时具有统计学意义。结果:流式细胞术检测BMM与PM细胞中F4-80标记的阳性巨噬细胞率分别约为96.6%和91.1%。一、IL-4刺激来源于WT与ASIC1a^-/-小鼠的BMM后:与WT相比,RT-PCR检测ASIC1a^-/-的M2型BMM标记物Arg1、FIZZ1、YM1、Mgl1和Mgl2 mRNA表达均出现不同程度的降低,WB检测Arg1蛋白表达明显下调;流式检测发现ASIC1a^-/-的M2型BMM表面分子CD206及胞内分子FIZZ1表达均明显下调。IL-4刺激WT与ASIC1a^-/-BMM不同时间点30min、60min、180min后,WB检测两组p-STAT6、STAT6表达水平之间无差异。二、IL-4刺激来源于WT与ASIC1a^-/-小鼠的组织巨噬细胞PM后:与WT相比,RT-PCR检测ASIC1a^-/-的M2型PM标记物Arg1、FIZZ1、YM1、Mgl1和Mgl2的mRNA表达也出现不同程度降低,WB检测Arg1蛋白表达下调;流式检测发现ASIC1a^-/-M2型PM表面分子CD206及胞内分子FIZZ1表达也均明显下调。IL-4刺激WT与ASIC1a^-/-的PM不同时间点30min、60min、180min后,WB检测p-STAT6、STAT6蛋白水平两组之间也无明显差异。结论:ASIC1a敲除引起M2型BMM与PM极化的下调,且此作用并非通过影响IL-4信号通路中的STAT6磷酸化下调M2型巨噬细胞的极化。更多还原

【Abstract】 Objective:Acid-sensitive ion channel（ASIC）is an H⁺-gated and Na⁺selective cation channel,which is the member of the epithelial sodium channel（ENaC）superfamily and could be activated by acidic environment.ASIC1s are expressed in the central nervous system,peripheral nervous system and nonneuronal cells such as dendritic cells（DCs）,macrophages,chondrocytes,osteoclasts,synoviocytes,pulmonary artery smooth muscle cells and so on.ASIC1 involves in multi-physiological and pathological processes,such as pain,learning,fear and ischemic stroke.Macrophages are widely considered important effector cells of the innate immune system.Depending on the phenotypes,macrophages are classified into the classically activated macrophages（M1）and alternatively activated macrophages（M2）.They play a vital role in the primary immune response related to pathogens,inflammation and tissue repair.Our previous study found that macrophage expresses functional ASIC1,M1 macrophages express higher membrane molecules CD80 and CD86 after ASIC1a knockout.It was suggested that ASIC1a may inhibit the antigen presentation of M1macrophages.The role of ASIC1a in the polarization process of M2 macrophages has not been studied.The aim of the present study are to investigate whether ASIC1a plays a role in the polarization of IL-4 induced mouse bone marrow-derived macrophage（BMM）and peritoneal macrophage（PM）into M2 phenotype,and discuss the mechanism of ASIC1a in M2 type polarization process.Material and Methods:BMM and PM were isolated from WT and ASIC1a^-/-mouse,the percentage of F4-80 positive macrophages were analyzed by flow cytometry assay.Then M2 macrophages were induced by IL-4.The M2 macrophage markers,Arg1,FIZZ1,YM1,Mgl1 and Mgl2 mRNA were detected by RT-PCR,and the specific protein Arg1 and ASIC1 were detected by Western blotting（WB）.The expression of M2 surface molecule CD206 and intracellular FIZZ1 were evaluated by flow cytometry.BMM and PM were induced by IL-4 after different time（30min/60min/180min）,the protein expression of STAT6 phosphorylation was detected by WB.All experiment data was reported as mean±standard deviation.GraphPad Prism 6 was used for statistical ananlysis.ANOVA,Student t test were used as indicated.Differences were considered at P﹤0.05.Results:The percentage of F4-80 positive macrophages in BMM/PM were 96.6%and91.1%.I.The BMM derived from WT and ASIC1a^-/-mouse:After induced by IL-4for 24 h,the gene expression of Arg1,FIZZ1,YM1,Mgl1 and Mgl2 in ASIC1a^-/-group were significantly lower than that in the WT group.And the protein expression of Arg1 were also down-regulated.The expression of surface CD206 and intracellular FIZZ1 of BMM in ASIC1a^-/-group were significantly lower than that in the WT group.After induced by IL-4 for different time（0min/30min/60min/180min）,the protein expression of p-STAT6/STAT6 in WT and ASIC1a^-/-group had no significant difference.Ⅱ.The PM derived from WT and ASIC1a^-/-mouse:After induced by IL-4for 24h,the gene expression of Arg1,FIZZ1,YM1,Mgl1 and Mgl2 and the protein expression of Arg1 in ASIC1a^-/-group were significantly lower than that in the WT group.The expression of surface CD206 and intracellular FIZZ1 of PM in ASIC1a^-/-group were significantly lower than that in the WT group.After induced by IL-4 for different time（30min/60min/180min）,the the protein expression of p-STAT6/STAT6in WT and ASIC1a^-/-group had no significant difference.Conclusions:ASIC1a knockout causes a significant down-regulation of BMM and PM polarization to M2 phenotype,and this effect was not caused by STAT6phosphorylation in the IL-4 signaling pathway.更多还原