本文選題：巨噬細(xì)胞 + 微環(huán)境酸化��； 參考：《華中科技大學(xué)》2011年博士論文

【摘要】：機(jī)體器官和組織的pH值一般維持在7.2～7.4。許多免疫相關(guān)的病理過(guò)程(如急性哮喘、腫瘤、炎癥和自身免疫病等)病灶組織局部會(huì)出現(xiàn)酸化,從而可能對(duì)免疫性疾病的發(fā)生產(chǎn)生重要影響。 巨噬細(xì)胞是機(jī)體最重要的免疫細(xì)胞之一,其病理生理學(xué)意義為：是機(jī)體清除病原微生物的第一道防線；可通過(guò)細(xì)胞間相互作用而調(diào)節(jié)固有免疫和適應(yīng)性免疫應(yīng)答；廣泛參與炎癥、腫瘤、自身免疫病等免疫相關(guān)疾病發(fā)生、發(fā)展。 酸敏感離子通道(acid-sensing ion channel, ASIC)屬上皮鈉通道/退化蛋白(epithelial sodium channel/degenerin, ENaC/DEC)家族成員,是H+門控的離子通道。目前已知,ASIC主要表達(dá)于中樞神經(jīng)系統(tǒng)和外周神經(jīng)元,參與痛、觸、味覺的形成以及學(xué)習(xí)記憶。近期文獻(xiàn)報(bào)道,T細(xì)胞、破骨細(xì)胞、平滑肌細(xì)胞和樹突狀細(xì)胞表面也可表達(dá)ASIC。 本課題主要探討微環(huán)境酸化對(duì)小鼠骨髓來(lái)源巨噬細(xì)胞(bone marrow derive macrophage, BMM)功能的影響,以及此作用與ASIC的相關(guān)性。 一、微環(huán)境酸化對(duì)BMM功能的影響 1.微環(huán)境酸化促進(jìn)BMM內(nèi)吞功能： (1)微環(huán)境酸化促進(jìn)BMM吞飲葡聚糖： 在酸性環(huán)境(pH 6.5)和生理環(huán)境(pH 7.3)下,使用FITC標(biāo)記的葡聚糖檢測(cè)BMM吞飲功能。結(jié)果發(fā)現(xiàn)：BMM吞飲能力與FITC-葡聚糖濃度呈正相關(guān),且酸性環(huán)境(pH6.5)可增強(qiáng)BMM吞飲作用。應(yīng)用FITC-葡聚糖(1000μg/ml)分析時(shí)間梯度,結(jié)果顯示：FITC-葡聚糖在BMM內(nèi)聚集呈時(shí)間依賴性,共孵育30 min后酸化組(pH 6.5)與生理組(pH 7.3)出現(xiàn)統(tǒng)計(jì)學(xué)差異(n=6, P0.05)。提示：微環(huán)境酸化促進(jìn)BMM吞飲FITC-葡聚糖。 (2)微環(huán)境酸化促進(jìn)BMM內(nèi)吞IgG包被乳膠顆粒： BMM內(nèi)吞葡聚糖主要依賴吞飲形式而實(shí)現(xiàn),故本課題應(yīng)用IgG包被乳膠顆粒進(jìn)一步觀察微環(huán)境酸化對(duì)IgG所介導(dǎo)內(nèi)吞的影響。結(jié)果顯示：BMM與IgG包被乳膠顆粒共孵育5 min,酸化組(pH 6.5)多數(shù)BMM內(nèi)吞6個(gè)以上顆粒,生理組(pH 7.3)多數(shù)BMM僅內(nèi)吞0～2個(gè)顆粒,且酸化組(pH 6.5)比生理組(pH 7.3)的吞噬指數(shù)高55.8%(n=6,P0.05)。以上結(jié)果提示：微環(huán)境酸化可增強(qiáng)BMM調(diào)理內(nèi)吞功能。 2.微環(huán)境酸化促進(jìn)BMM表面CD80、CD86、MHC 11分子表達(dá) 將生理狀態(tài)下(pH 7.3,5% CO2)誘導(dǎo)分化的BMM置于酸化環(huán)境(pH 6.5培養(yǎng)基)培養(yǎng)3 h(37℃,7% CO2),流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示：微環(huán)境酸化可上調(diào)BMM表面CD80、CD86、MHCⅡ表達(dá)。 3.微環(huán)境酸化促進(jìn)BMM分泌IL-10,但對(duì)BMM分泌TNF-a無(wú)影響 BMM分別在生理環(huán)境(pH 7.3,5% CO2)和酸化環(huán)境(pH 6.5,7% CO2)培養(yǎng)3h,采集培養(yǎng)上清,離心去除細(xì)胞碎片,-80℃保存,分別檢測(cè)IL-10和TNF-α水平。結(jié)果顯示：酸化環(huán)境可刺激BMM分泌IL-10,但對(duì)TNF-α分泌無(wú)影響。 二、微環(huán)境酸化調(diào)控BMM功能與ASIC相關(guān) 1.BMM表達(dá)ASIC 采集誘導(dǎo)分化的BMM,提取細(xì)胞總RNA和總蛋白,借助RT-PCR和Western blot分別檢測(cè)ASIC mRNA和蛋白表達(dá)；借助細(xì)胞免疫熒光技術(shù)檢測(cè)BMM內(nèi)ASIC分布。結(jié)果顯示：ASIC1和ASIC3主要表達(dá)于BMM胞質(zhì)中,BMM不表達(dá)ASIC2。 2.微環(huán)境酸化促進(jìn)BMM吞飲葡聚糖與ASIC相關(guān) 應(yīng)用ASIC阻斷劑阿米洛利(amiloride,100μM)探討ASIC是否參與微環(huán)境酸化促進(jìn)BMM吞飲葡聚糖的作用,結(jié)果發(fā)現(xiàn)：在pH 7.3培養(yǎng)基中,阿米洛利預(yù)處理BMM30 min,其對(duì)生理組BMM(pH 7.3)吞飲FITC-葡聚糖無(wú)影響(n=6,P0.05)；而阿米洛利預(yù)處理可明顯抑制酸化組(pH 6.5)BMM吞飲作用(n=6,P0.05)。提示：微環(huán)境酸化上調(diào)BMM吞飲功能與ASIC相關(guān)。 3.微環(huán)境酸化上調(diào)BMM相關(guān)膜分子的表達(dá)與ASIC相關(guān) 應(yīng)用阿米洛利(100μM),探討微環(huán)境酸化上調(diào)BMM膜分子表達(dá)與ASIC的相關(guān)性。生理組(pH 7.3)用阿米洛利預(yù)處理BMM 30 min(37℃,5% CO2),然后換用pH6.5的培養(yǎng)基培養(yǎng)3 h(37℃,7% CO2),通過(guò)流式細(xì)胞術(shù)檢測(cè)CD80、CD86、MHC 11分子表達(dá)。結(jié)果顯示：酸化阻斷組(pH 6.5+阿米洛利)與單純酸化組(pH 6.5)組相比,上述膜分子表達(dá)明顯下調(diào)(n=6,P0.05),而與生理組(pH 7.3)無(wú)顯著差異(n=6, P0.05)。提示：微環(huán)境酸化上調(diào)BMM膜分子表達(dá)的作用與ASIC相關(guān)。 4.非甾體抗炎藥可抑制微環(huán)境酸化對(duì)BMM膜分子表達(dá)的上調(diào) 文獻(xiàn)報(bào)道,非甾體抗炎藥(non-steroidal anti-inflammatory drug, NSAID)可抑制多種神經(jīng)元ASIC電流。本研究應(yīng)用NSAID分析BMM表面ASIC的功能。應(yīng)用布洛芬(ibuprofen,200μM)或雙氯芬酸(diclofenac,200μM)預(yù)處理BMM(pH 7.3,5% CO2,30 min),然后酸化組換用pH 6.5的培養(yǎng)基培養(yǎng)3h(37℃,7% CO2),流式細(xì)胞術(shù)檢測(cè)CD80、CD86、MHCⅡ表達(dá)。結(jié)果顯示：NSAID預(yù)處理后,酸化組BMM膜分子表達(dá)明顯下調(diào)(n=6,P0.05),而生理組BMM無(wú)明顯改變(n=6,P0.05)。提示：微環(huán)境酸化上調(diào)BMM膜分子表達(dá)的作用與ASIC相關(guān)。 結(jié)論 1.微環(huán)境酸化可促進(jìn)巨噬細(xì)胞的吞噬功能、膜分子表達(dá)和分泌IL-10； 2.BMM表達(dá)ASIC1和ASIC3； 3.微環(huán)境酸化對(duì)BMM功能的調(diào)控作用與ASIC相關(guān)。
[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，

本文編號(hào)：1918432