本文關(guān)鍵詞： 線蟲表皮細(xì)胞 結(jié)構(gòu)破壞 抗菌肽 線蟲半橋粒 STA-2　出處：《蘇州大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：表皮細(xì)胞層作為一道物理屏障經(jīng)常遭受各種破壞其結(jié)構(gòu)的物理損傷。但是表皮細(xì)胞免疫系統(tǒng)是否能夠?qū)⒔Y(jié)構(gòu)的破壞作為一種危險(xiǎn)信號(hào)來激發(fā)免疫應(yīng)答還不清楚。由于哺乳動(dòng)物表皮細(xì)胞層結(jié)構(gòu)復(fù)雜,包含多種免疫細(xì)胞的干擾。而模式生物秀麗隱桿線蟲表皮細(xì)胞層結(jié)構(gòu)簡(jiǎn)單,主要由一個(gè)單層巨大多核上皮細(xì)胞組成,是研究表皮細(xì)胞自身免疫功能的完美模型。所以我們利用線蟲表皮上皮細(xì)胞為模型來研究這一科學(xué)問題。線蟲表皮細(xì)胞在遭到穿透性感染或者機(jī)械損傷后會(huì)激發(fā)表皮細(xì)胞免疫防御引起抗菌肽的表達(dá)釋放。通過系統(tǒng)性的敲除線蟲表皮細(xì)胞各支撐結(jié)構(gòu)組成蛋白,我們發(fā)現(xiàn)只有頂部半橋粒的破壞能夠顯著的激發(fā)表皮細(xì)胞固有免疫應(yīng)答,引起抗菌肽的轉(zhuǎn)錄上調(diào)。線蟲表皮細(xì)胞通過半橋粒頂部蛋白MUP-4以及粘附在其上的STAT家族蛋白STA-2來識(shí)別結(jié)構(gòu)破壞。當(dāng)表皮細(xì)胞嚴(yán)重受損致使頂部半橋粒解體時(shí),STA-2便會(huì)從頂部半橋粒上脫落引發(fā)下游抗菌肽的轉(zhuǎn)錄上調(diào)。這樣的免疫應(yīng)答機(jī)制使得表皮細(xì)胞在遭受大規(guī)模結(jié)構(gòu)破壞時(shí)可以直接啟動(dòng)抗菌肽的表達(dá)而不需要通過一步步的信號(hào)傳導(dǎo)過程。同時(shí)我們利用人類表皮角質(zhì)層細(xì)胞將在線蟲表皮細(xì)胞中的發(fā)現(xiàn)延伸到了哺乳動(dòng)物細(xì)胞中。我們的研究揭示了一種進(jìn)化上保守的上皮細(xì)胞識(shí)別危險(xiǎn)以及激活免疫應(yīng)答的分子機(jī)制。下面從實(shí)驗(yàn)?zāi)康�、方法、結(jié)果、結(jié)論和關(guān)鍵詞幾方面來簡(jiǎn)要總結(jié)我們的研究?jī)?nèi)容。目的:1.尋找能夠引起線蟲表皮細(xì)胞固有免疫應(yīng)答的結(jié)構(gòu)原件。2.闡述結(jié)構(gòu)破壞引起的線蟲表皮細(xì)胞免疫應(yīng)答的分子機(jī)制。3.探討頂部半橋粒解體引發(fā)的線蟲表皮細(xì)胞固有免疫應(yīng)答的生理意義。4.檢測(cè)我們?cè)诰�蟲中的發(fā)現(xiàn)是否同樣存在于哺乳動(dòng)物細(xì)胞中。方法:1,a)利用免疫熒光染色來詳細(xì)記錄秀麗線蟲晚期幼蟲負(fù)責(zé)維持表皮細(xì)胞形態(tài)結(jié)構(gòu)穩(wěn)定的各類支撐結(jié)構(gòu)。b)利用RNAi或者藥物處理的方法來敲降表皮細(xì)胞各結(jié)構(gòu)蛋白,檢測(cè)對(duì)抗菌肽上調(diào)的影響。2,a)利用RNAi和蛋白功能缺失的突變體蟲株來逐個(gè)檢測(cè)半橋粒成分缺失后線蟲表皮細(xì)胞固有免疫應(yīng)答的激發(fā)情況。b)利用線蟲表皮細(xì)胞調(diào)控固有免疫應(yīng)答的各個(gè)信號(hào)通路成分的突變體蟲株來查找介導(dǎo)頂部半橋粒解體引起的固有免疫應(yīng)答的信號(hào)通路。c)利用STAT家族蛋白STA-2和半橋粒成員的免疫熒光雙染觀測(cè)內(nèi)源STA-2在線蟲表皮細(xì)胞的亞細(xì)胞定位以及STA-2和半橋粒的空間位置關(guān)系;同時(shí)利用免疫共沉淀技術(shù)檢測(cè)STA-2和半橋粒頂部蛋白MUP-4是否屬于同一個(gè)蛋白復(fù)合體,MUP-4是線蟲表皮細(xì)胞結(jié)構(gòu)破壞引發(fā)固有免疫應(yīng)答中的一個(gè)關(guān)鍵蛋白。3,a)我們發(fā)展了一種使用單層微米級(jí)的玻璃碎片的損傷新方法,這種方法可以一次在多條線蟲表皮層造成多個(gè)傷口。b)對(duì)野生型、p38MAPK信號(hào)通路和STA-2的突變體蟲株中各自進(jìn)行兩種方法的損傷,以此來檢測(cè)表皮細(xì)胞多個(gè)傷口和單個(gè)傷口引起的免疫應(yīng)答途徑是否有所不同。4,a)我們?cè)隗w外培養(yǎng)的成年人類表皮角質(zhì)層細(xì)胞里檢測(cè)了五種支撐結(jié)構(gòu)破壞后三種抗菌肽和三種細(xì)胞因子的表達(dá)。b)利用si RNA和抑制劑的方法分別檢測(cè)了哺乳動(dòng)物中7個(gè)STAT轉(zhuǎn)錄因子、p38MAPK和NF-k B這兩條信號(hào)通路在表皮細(xì)胞結(jié)構(gòu)破壞引發(fā)的固有免疫應(yīng)答的參與情況。結(jié)果:1,a)免疫熒光雙染結(jié)果顯示線蟲表皮細(xì)胞大多數(shù)支撐結(jié)構(gòu)排列有序,彼此之間很少交聯(lián)。b)通過觀察表皮細(xì)胞免疫應(yīng)答標(biāo)志基因nlp-29啟動(dòng)子控制的GFP表達(dá)變化,發(fā)現(xiàn)大部分幫助支撐表皮細(xì)胞的結(jié)構(gòu)缺失后并不會(huì)引起表皮細(xì)胞抗菌肽轉(zhuǎn)錄上調(diào)。只有半橋粒頂膜受體mup-4和外皮膠原蛋白bli-1的敲除引發(fā)了顯著的抗菌肽轉(zhuǎn)錄上調(diào)。半橋粒核心蛋白vab-10a和中間纖維ifb-1的敲除也引發(fā)了適量抗菌肽轉(zhuǎn)錄上調(diào)。2,a)和半橋粒的頂膜受體mup-4相比,其它半橋粒成分的缺失并未顯著上調(diào)表皮細(xì)胞抗菌肽。b)線蟲表皮細(xì)胞調(diào)控免疫應(yīng)答的各個(gè)信號(hào)通路成分的突變體蟲株都不能阻斷頂部半橋粒解體引起的抗菌肽上調(diào),只有一個(gè)STAT家族蛋白STA-2的突變體可以阻斷這一免疫應(yīng)答。c)免疫熒光染色結(jié)果顯示STA-2在表皮細(xì)胞里呈現(xiàn)出經(jīng)典的半橋粒樣的條帶并且STA-2和半橋粒共定位在一起;免疫共沉淀結(jié)果表明MUP-4和STA-2在線蟲表皮細(xì)胞中是以同一個(gè)蛋白復(fù)合體的形式存在。3,a)通過對(duì)鬼筆環(huán)肽標(biāo)記傷口照片的觀察發(fā)現(xiàn),相比舊方法,玻璃碎片的損傷方法確實(shí)可以給線蟲表皮細(xì)胞一次造成多個(gè)傷口。b)表皮細(xì)胞單一傷口激發(fā)的免疫應(yīng)答需要經(jīng)由p38MAPK信號(hào)通路和STA-2,但多個(gè)傷口引發(fā)的免疫應(yīng)答不再通過p38MAPK信號(hào)級(jí)聯(lián),只有STA-2的功能是必需的。4,a)人類表皮角質(zhì)層細(xì)胞中肌動(dòng)蛋白(微絲)、微管、角蛋白(中間纖維)和黏著斑這四種細(xì)胞支撐結(jié)構(gòu)破壞并不會(huì)激發(fā)顯著的固有免疫應(yīng)答,只有半橋粒蛋白復(fù)合體的破壞會(huì)激發(fā)顯著的固有免疫應(yīng)答。b)七個(gè)STAT中只有STAT3和STAT5B可以減弱由半橋粒蛋白復(fù)合體破壞引發(fā)的固有免疫應(yīng)答;p38 MAPK和NF-k B這兩條信號(hào)通路并不參與調(diào)節(jié)半橋粒蛋白復(fù)合體破壞引發(fā)的固有免疫應(yīng)答。結(jié)論:1.線蟲表皮細(xì)胞大部分支撐結(jié)構(gòu)的空間構(gòu)架具有高度規(guī)律性并區(qū)域分明。2.能夠引發(fā)線蟲表皮細(xì)胞固有免疫應(yīng)答的支撐結(jié)構(gòu)具有空間特異性,聚集在頂部半橋粒附近。3.半橋粒頂膜受體MUP-4是介導(dǎo)表皮細(xì)胞結(jié)構(gòu)破壞引發(fā)的固有免疫應(yīng)答的關(guān)鍵蛋白。4.半橋粒破壞引發(fā)的固有免疫應(yīng)答需要STAT家族蛋白STA-2,但是不需要表皮細(xì)胞其它的免疫相關(guān)信號(hào)通路。5.STA-2和半橋粒共定位于線蟲表皮細(xì)胞的頂膜。6.線蟲表皮細(xì)胞大規(guī)模損傷激發(fā)的固有免疫應(yīng)答會(huì)繞過p38 MAPK信號(hào)通路而只依賴于STA-2。7.在成年人類表皮角質(zhì)層細(xì)胞中,半橋粒蛋白復(fù)合體的破壞能夠激發(fā)顯著的固有免疫應(yīng)答,并且這一免疫應(yīng)答是需要轉(zhuǎn)錄因子STAT3和STAT5B的功能。
[Abstract]:The epidermal cell layer as a physical barrier often suffer from physical damage. But the structure of various epidermal cell immune system would destroy the structure as a danger signal to stimulate the immune response is not clear. Because mammalian cells in the epidermis of complex structure, contains a variety of immune cells and interference. The model organism Caenorhabditis elegans the epidermal cell layer has the advantages of simple structure, mainly composed of a single large multinucleated epithelial cells, is a perfect model of epidermal cell immune function. So we use the nematode epidermal epithelial cells as model to study the scientific problems. By penetrating the epidermal cells in nematode infection or after mechanical injury can stimulate epidermal cell immune defense the release caused by expression of antimicrobial peptide. Through systematic knockout nematode epidermal cells of each of the support structures composed of protein, we found that only The top of hemidesmosomes damage can significantly stimulate epidermal cell innate immune responses caused by increased transcription of antimicrobial peptides. The nematode epidermal cells by hemidesmosome top protein MUP-4 and adhesion on the STAT family protein STA-2 to identify the structural damage. When the epidermal cells were damaged severely in the top of hemidesmosome disintegration, STA-2 will fall from the top hemidesmosomes caused increased transcription of downstream antimicrobial peptides. The immune response mechanism of epidermal cells can directly initiate the expression of antimicrobial peptides without through signal transduction process step by step in suffered massive when the structure is damaged. At the same time we use human epidermal cuticle cells in the epidermal cells of C.elegans found extended to mammals the molecular mechanism of the cell. Our study reveals an evolutionarily conserved epithelial cell recognition of risk and activation of immune responses. The following from the objective, methods, results, several conclusions and keywords to a brief summary of our research content. Objective: to find the 1. nematode can cause epidermal cells of the innate immune response of.3..2. structure original molecular mechanism of structure damage nematode epidermal cells induced immune response to.4. at the top of the physiological significance of hemidesmosome disintegration of epidermal cell intrinsic nematode the immune response triggered by the detection of insects found in our online is also exist in mammalian cells. Methods: 1, a) by using immunofluorescence staining with records of Caenorhabditis elegans late larval epidermal cells responsible for maintaining all kinds of morphological structure and stable support structure.B) method using RNAi or drug treatment to knock down the epidermal cell structure protein, to detect the influence of cathelicidin upregulation of.2, RNAi and a) using protein deficient mutant strains by detecting half desmosomes Stimulate.B nematode epidermal cells of the innate immune response after each ingredient) signaling pathway components of the innate immune response by nematode epidermal cells mutant strains to find the top of hemidesmosome mediated innate immune response caused by the disintegration of the signaling pathway.C) using STAT family protein STA-2 and hemidesmosome member subcellular immunofluorescence double staining observation of endogenous STA-2 nematode epidermal cells and STA-2 and hemidesmosome spatial relationship; at the same time using the immune is belong to the same protein complex co precipitation detection STA-2 and hemidesmosome top protein MUP-4 technology, MUP-4 is a key protein.3 triggered innate immune response in the destruction of the structure of nematode epidermal cells, we develop a) a new method of using a single damage micron glass fragments, this method can be a plurality in the epidermal layer caused multiple nematode The wound of.B) of the wild type, the p38MAPK signal transduction pathway and STA-2 mutant strains in each of the two methods of damage, immune response way to detect epidermal cells of multiple wounds and single wound caused by different.4, a) of adult human epidermal cuticle cells we cultured in vitro in the detection of the expression of.B five kinds of support structures after the destruction of three kinds of antibacterial peptides and three kinds of cytokines) were detected in 7 STAT transcription factor in mammals by using the method of Si and RNA inhibitors, in the innate immune response of p38MAPK and NF-k B of the two signal pathways caused by the destruction of epidermal cell structure. Results: 1, a) double immunofluorescence staining showed that epidermal cells arranged in nematode support structure most orderly, each other rarely cross-linked.B) marked by observation of epidermal cell immune response gene nlp-29 promoter to control the expression of GFP variant Help, found that most of the epidermal cells after lack of support structure does not cause antibacterial peptide increased transcription of epidermal cells. Only hemidesmosome apical membrane receptor mup-4 and skin collagen bli-1 knockdown caused significant transcriptional upregulation. Antibacterial peptide hemidesmosome core protein vab-10a and ifb-1 in fiber knockout also led to the amount of antimicrobial peptides the increased transcription of.2, a) compared with the hemidesmosomes of apical membrane receptor mup-4, antibacterial peptide.B lack of other components did not significantly increase hemidesmosome epidermal cells) each pathway components of nematode epidermal cell immune response of the mutant strains are not blocking the top hemidesmosomes caused disintegration of antibacterial peptide increases, only one STAT mutants of STA-2 family proteins can inhibit the immune response of.C) immunofluorescence staining showed that STA-2 in epidermal cells showed the classic hemidesmosome kind of strip and ST A-2 and hemidesmosome co localization together; CO immunoprecipitation results showed that MUP-4 and STA-2 worms in epidermal cells is a protein complex with the form of.3, a) through the observation of phalloidin labeled wound photos found, compared to the old method, the damage method of broken glass can indeed cause multiple wounds.B to the epidermal cells of a nematode) immune response to epidermal cells single wound excitation via p38MAPK and STA-2 signaling pathways, but the immune response to multiple wounds caused no longer through the p38MAPK signaling cascade, only the STA-2 function is required in.4, a) of human epidermal cuticle cells (actin microfilaments, microtubules, keratin) (intermediate filament) and focal adhesion of the four cell support structure damage does not significantly stimulate the innate immune response, only hemidesmosome protein complex damage can stimulate the innate immune response of.B was seven) STAT only STAT3 and STAT5B can weaken the innate immune response caused by destruction of hemidesmosome protein complex; p38 MAPK and NF-k B of the two signal pathways are not involved in the regulation of hemidesmosome protein complex destruction of innate immune response triggered. Conclusion: the supporting structure of the 1. nematode epidermal cell support structure most space frame with a high degree of regularity and regional clearly.2. is able to induce nematode epidermal cells of the innate immune response is specific, gathered at the top of hemidesmosomes near.3. hemidesmosome apical membrane receptor MUP-4 is the innate immune response mediated by epidermal cell damage caused by the innate immune response of.4. protein hemidesmosome damage caused by the need of STAT family protein STA-2, but not epidermal cells other immune related.5.STA-2 signaling pathway and hemidesmosomes total epidermal cells located in the apical membrane of.6. elegans nematode epidermal cells The innate immune response of large-scale damage excitation will bypass the p38 MAPK signal pathway depends only on STA-2.7. in adult human epidermal cuticle cells, hemidesmosome complex protein damage can stimulate innate immune response significantly, and the immune response is to transcription factors STAT3 and STAT5B function.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392

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