微环境酸化影响BMM的功能及此效应与ASIC的相关性

发布时间：2018-05-21 08:14

  本文选题：巨噬细胞 + 微环境酸化　； 参考：《华中科技大学》2011年博士论文

【摘要】：机体器官和组织的pH值一般维持在7.2～7.4。许多免疫相关的病理过程(如急性哮喘、肿瘤、炎症和自身免疫病等)病灶组织局部会出现酸化,从而可能对免疫性疾病的发生产生重要影响。 巨噬细胞是机体最重要的免疫细胞之一,其病理生理学意义为：是机体清除病原微生物的第一道防线；可通过细胞间相互作用而调节固有免疫和适应性免疫应答；广泛参与炎症、肿瘤、自身免疫病等免疫相关疾病发生、发展。 酸敏感离子通道(acid-sensing ion channel, ASIC)属上皮钠通道/退化蛋白(epithelial sodium channel/degenerin, ENaC/DEC)家族成员,是H+门控的离子通道。目前已知,ASIC主要表达于中枢神经系统和外周神经元,参与痛、触、味觉的形成以及学习记忆。近期文献报道,T细胞、破骨细胞、平滑肌细胞和树突状细胞表面也可表达ASIC。 本课题主要探讨微环境酸化对小鼠骨髓来源巨噬细胞(bone marrow derive macrophage, BMM)功能的影响,以及此作用与ASIC的相关性。 一、微环境酸化对BMM功能的影响 1.微环境酸化促进BMM内吞功能： (1)微环境酸化促进BMM吞饮葡聚糖： 在酸性环境(pH 6.5)和生理环境(pH 7.3)下,使用FITC标记的葡聚糖检测BMM吞饮功能。结果发现：BMM吞饮能力与FITC-葡聚糖浓度呈正相关,且酸性环境(pH6.5)可增强BMM吞饮作用。应用FITC-葡聚糖(1000μg/ml)分析时间梯度,结果显示：FITC-葡聚糖在BMM内聚集呈时间依赖性,共孵育30 min后酸化组(pH 6.5)与生理组(pH 7.3)出现统计学差异(n=6, P0.05)。提示：微环境酸化促进BMM吞饮FITC-葡聚糖。 (2)微环境酸化促进BMM内吞IgG包被乳胶颗粒： BMM内吞葡聚糖主要依赖吞饮形式而实现,故本课题应用IgG包被乳胶颗粒进一步观察微环境酸化对IgG所介导内吞的影响。结果显示：BMM与IgG包被乳胶颗粒共孵育5 min,酸化组(pH 6.5)多数BMM内吞6个以上颗粒,生理组(pH 7.3)多数BMM仅内吞0～2个颗粒,且酸化组(pH 6.5)比生理组(pH 7.3)的吞噬指数高55.8%(n=6,P0.05)。以上结果提示：微环境酸化可增强BMM调理内吞功能。 2.微环境酸化促进BMM表面CD80、CD86、MHC 11分子表达 将生理状态下(pH 7.3,5% CO2)诱导分化的BMM置于酸化环境(pH 6.5培养基)培养3 h(37℃,7% CO2),流式细胞术检测结果显示：微环境酸化可上调BMM表面CD80、CD86、MHCⅡ表达。 3.微环境酸化促进BMM分泌IL-10,但对BMM分泌TNF-a无影响 BMM分别在生理环境(pH 7.3,5% CO2)和酸化环境(pH 6.5,7% CO2)培养3h,采集培养上清,离心去除细胞碎片,-80℃保存,分别检测IL-10和TNF-α水平。结果显示：酸化环境可刺激BMM分泌IL-10,但对TNF-α分泌无影响。 二、微环境酸化调控BMM功能与ASIC相关 1.BMM表达ASIC 采集诱导分化的BMM,提取细胞总RNA和总蛋白,借助RT-PCR和Western blot分别检测ASIC mRNA和蛋白表达；借助细胞免疫荧光技术检测BMM内ASIC分布。结果显示：ASIC1和ASIC3主要表达于BMM胞质中,BMM不表达ASIC2。 2.微环境酸化促进BMM吞饮葡聚糖与ASIC相关 应用ASIC阻断剂阿米洛利(amiloride,100μM)探讨ASIC是否参与微环境酸化促进BMM吞饮葡聚糖的作用,结果发现：在pH 7.3培养基中,阿米洛利预处理BMM30 min,其对生理组BMM(pH 7.3)吞饮FITC-葡聚糖无影响(n=6,P0.05)；而阿米洛利预处理可明显抑制酸化组(pH 6.5)BMM吞饮作用(n=6,P0.05)。提示：微环境酸化上调BMM吞饮功能与ASIC相关。 3.微环境酸化上调BMM相关膜分子的表达与ASIC相关 应用阿米洛利(100μM),探讨微环境酸化上调BMM膜分子表达与ASIC的相关性。生理组(pH 7.3)用阿米洛利预处理BMM 30 min(37℃,5% CO2),然后换用pH6.5的培养基培养3 h(37℃,7% CO2),通过流式细胞术检测CD80、CD86、MHC 11分子表达。结果显示：酸化阻断组(pH 6.5+阿米洛利)与单纯酸化组(pH 6.5)组相比,上述膜分子表达明显下调(n=6,P0.05),而与生理组(pH 7.3)无显著差异(n=6, P0.05)。提示：微环境酸化上调BMM膜分子表达的作用与ASIC相关。 4.非甾体抗炎药可抑制微环境酸化对BMM膜分子表达的上调 文献报道,非甾体抗炎药(non-steroidal anti-inflammatory drug, NSAID)可抑制多种神经元ASIC电流。本研究应用NSAID分析BMM表面ASIC的功能。应用布洛芬(ibuprofen,200μM)或双氯芬酸(diclofenac,200μM)预处理BMM(pH 7.3,5% CO2,30 min),然后酸化组换用pH 6.5的培养基培养3h(37℃,7% CO2),流式细胞术检测CD80、CD86、MHCⅡ表达。结果显示：NSAID预处理后,酸化组BMM膜分子表达明显下调(n=6,P0.05),而生理组BMM无明显改变(n=6,P0.05)。提示：微环境酸化上调BMM膜分子表达的作用与ASIC相关。 结论 1.微环境酸化可促进巨噬细胞的吞噬功能、膜分子表达和分泌IL-10； 2.BMM表达ASIC1和ASIC3； 3.微环境酸化对BMM功能的调控作用与ASIC相关。
[Abstract]:The pH values of organs and tissues of the organism are generally maintained at 7.2 - 7.4 . Many immune - related pathological processes ( such as acute asthma , tumor , inflammation and autoimmune diseases ) can be acidified locally , which may have an important effect on the occurrence of immune diseases .


The macrophages are one of the most important immune cells in the organism , and the physiopathological significance is : the first line of defense of the organism to clear the pathogenic microorganism ;
the innate immune and adaptive immune responses can be adjusted by inter - cell interaction ;
can be extensively involved in the development of immune - related diseases such as inflammation , tumor , autoimmune disease and the like .


The acid - sensing ion channel ( ASIC ) belongs to the family member of epithelial sodium channel / retreat protein ( ENaC / DEC ) family and is an ion channel of H + gated ion . It is known that the ASIC is mainly expressed in central nervous system and peripheral neurons , participates in the formation of pain , touch , taste and learning and memory . Recent literature reports that the surface of T cell , Osteoclasts , smooth muscle cells and dendritic cells can also express ASIC .


The purpose of this study was to study the effect of micro - environmental acidification on bone marrow derived macrophages ( BMM ) and its correlation with ASIC .


Influence of One - and Micro - environment Acidification on BMM Function


1 . Micro - environment acidification promotes BMM endocytosis :


( 1 ) micro - environment acidification promotes BMM endocytosis :


The results showed that the concentration of FITC - dextran was positively correlated with the concentration of FITC - dextran , and the acidic environment ( pH6.5 ) could enhance BMM endocytosis . The results showed that the concentration of FITC - dextran in BMM was time - dependent . The results showed that the concentration of FITC - dextran in BMM was time - dependent , and the pH 6.5 and pH 7.3 showed statistical difference after 30 min incubation ( n = 6 , P0.05 ) . Tip : Micro - environment acidification promotes BMM endocytosis FITC - glucan .


( 2 ) micro - environment acidification promotes BMM endocytosis IgG coated latex particles :


The results showed that the BMM and IgG coated latex particles were incubated for 5 min , the pH was 6.5 , the majority of BMM contained only 0 - 2 granules , and the pH 6.5 was 55.8 % higher than that of the physiological group ( pH 7.3 ) ( n = 6 , P0.05 ) . The results suggested that micro - environment acidification could enhance BMM ' s endocytosis .


2 . Micro - environment Acidification promotes the expression of CD80 , CD86 and MHC 11 molecules on the surface of BMM


Under physiological condition ( pH 7.5 , 5 % CO2 ) , the differentiated BMM was cultured for 3 h ( pH 6.5 medium ) for 3 h ( 37 鈩，

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