STA-2介导的秀丽隐杆线虫表皮细胞结构破坏引发的固有免疫应答

发布时间：2018-01-20 22:20

本文关键词： 线虫表皮细胞结构破坏抗菌肽线虫半桥粒 STA-2　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：表皮细胞层作为一道物理屏障经常遭受各种破坏其结构的物理损伤。但是表皮细胞免疫系统是否能够将结构的破坏作为一种危险信号来激发免疫应答还不清楚。由于哺乳动物表皮细胞层结构复杂,包含多种免疫细胞的干扰。而模式生物秀丽隐杆线虫表皮细胞层结构简单,主要由一个单层巨大多核上皮细胞组成,是研究表皮细胞自身免疫功能的完美模型。所以我们利用线虫表皮上皮细胞为模型来研究这一科学问题。线虫表皮细胞在遭到穿透性感染或者机械损伤后会激发表皮细胞免疫防御引起抗菌肽的表达释放。通过系统性的敲除线虫表皮细胞各支撑结构组成蛋白,我们发现只有顶部半桥粒的破坏能够显著的激发表皮细胞固有免疫应答,引起抗菌肽的转录上调。线虫表皮细胞通过半桥粒顶部蛋白MUP-4以及粘附在其上的STAT家族蛋白STA-2来识别结构破坏。当表皮细胞严重受损致使顶部半桥粒解体时,STA-2便会从顶部半桥粒上脱落引发下游抗菌肽的转录上调。这样的免疫应答机制使得表皮细胞在遭受大规模结构破坏时可以直接启动抗菌肽的表达而不需要通过一步步的信号传导过程。同时我们利用人类表皮角质层细胞将在线虫表皮细胞中的发现延伸到了哺乳动物细胞中。我们的研究揭示了一种进化上保守的上皮细胞识别危险以及激活免疫应答的分子机制。下面从实验目的、方法、结果、结论和关键词几方面来简要总结我们的研究内容。目的:1.寻找能够引起线虫表皮细胞固有免疫应答的结构原件。2.阐述结构破坏引起的线虫表皮细胞免疫应答的分子机制。3.探讨顶部半桥粒解体引发的线虫表皮细胞固有免疫应答的生理意义。4.检测我们在线虫中的发现是否同样存在于哺乳动物细胞中。方法:1,a)利用免疫荧光染色来详细记录秀丽线虫晚期幼虫负责维持表皮细胞形态结构稳定的各类支撑结构。b)利用RNAi或者药物处理的方法来敲降表皮细胞各结构蛋白,检测对抗菌肽上调的影响。2,a)利用RNAi和蛋白功能缺失的突变体虫株来逐个检测半桥粒成分缺失后线虫表皮细胞固有免疫应答的激发情况。b)利用线虫表皮细胞调控固有免疫应答的各个信号通路成分的突变体虫株来查找介导顶部半桥粒解体引起的固有免疫应答的信号通路。c)利用STAT家族蛋白STA-2和半桥粒成员的免疫荧光双染观测内源STA-2在线虫表皮细胞的亚细胞定位以及STA-2和半桥粒的空间位置关系;同时利用免疫共沉淀技术检测STA-2和半桥粒顶部蛋白MUP-4是否属于同一个蛋白复合体,MUP-4是线虫表皮细胞结构破坏引发固有免疫应答中的一个关键蛋白。3,a)我们发展了一种使用单层微米级的玻璃碎片的损伤新方法,这种方法可以一次在多条线虫表皮层造成多个伤口。b)对野生型、p38MAPK信号通路和STA-2的突变体虫株中各自进行两种方法的损伤,以此来检测表皮细胞多个伤口和单个伤口引起的免疫应答途径是否有所不同。4,a)我们在体外培养的成年人类表皮角质层细胞里检测了五种支撑结构破坏后三种抗菌肽和三种细胞因子的表达。b)利用si RNA和抑制剂的方法分别检测了哺乳动物中7个STAT转录因子、p38MAPK和NF-k B这两条信号通路在表皮细胞结构破坏引发的固有免疫应答的参与情况。结果:1,a)免疫荧光双染结果显示线虫表皮细胞大多数支撑结构排列有序,彼此之间很少交联。b)通过观察表皮细胞免疫应答标志基因nlp-29启动子控制的GFP表达变化,发现大部分帮助支撑表皮细胞的结构缺失后并不会引起表皮细胞抗菌肽转录上调。只有半桥粒顶膜受体mup-4和外皮胶原蛋白bli-1的敲除引发了显著的抗菌肽转录上调。半桥粒核心蛋白vab-10a和中间纤维ifb-1的敲除也引发了适量抗菌肽转录上调。2,a)和半桥粒的顶膜受体mup-4相比,其它半桥粒成分的缺失并未显著上调表皮细胞抗菌肽。b)线虫表皮细胞调控免疫应答的各个信号通路成分的突变体虫株都不能阻断顶部半桥粒解体引起的抗菌肽上调,只有一个STAT家族蛋白STA-2的突变体可以阻断这一免疫应答。c)免疫荧光染色结果显示STA-2在表皮细胞里呈现出经典的半桥粒样的条带并且STA-2和半桥粒共定位在一起;免疫共沉淀结果表明MUP-4和STA-2在线虫表皮细胞中是以同一个蛋白复合体的形式存在。3,a)通过对鬼笔环肽标记伤口照片的观察发现,相比旧方法,玻璃碎片的损伤方法确实可以给线虫表皮细胞一次造成多个伤口。b)表皮细胞单一伤口激发的免疫应答需要经由p38MAPK信号通路和STA-2,但多个伤口引发的免疫应答不再通过p38MAPK信号级联,只有STA-2的功能是必需的。4,a)人类表皮角质层细胞中肌动蛋白(微丝)、微管、角蛋白(中间纤维)和黏着斑这四种细胞支撑结构破坏并不会激发显著的固有免疫应答,只有半桥粒蛋白复合体的破坏会激发显著的固有免疫应答。b)七个STAT中只有STAT3和STAT5B可以减弱由半桥粒蛋白复合体破坏引发的固有免疫应答;p38 MAPK和NF-k B这两条信号通路并不参与调节半桥粒蛋白复合体破坏引发的固有免疫应答。结论:1.线虫表皮细胞大部分支撑结构的空间构架具有高度规律性并区域分明。2.能够引发线虫表皮细胞固有免疫应答的支撑结构具有空间特异性,聚集在顶部半桥粒附近。3.半桥粒顶膜受体MUP-4是介导表皮细胞结构破坏引发的固有免疫应答的关键蛋白。4.半桥粒破坏引发的固有免疫应答需要STAT家族蛋白STA-2,但是不需要表皮细胞其它的免疫相关信号通路。5.STA-2和半桥粒共定位于线虫表皮细胞的顶膜。6.线虫表皮细胞大规模损伤激发的固有免疫应答会绕过p38 MAPK信号通路而只依赖于STA-2。7.在成年人类表皮角质层细胞中,半桥粒蛋白复合体的破坏能够激发显著的固有免疫应答,并且这一免疫应答是需要转录因子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.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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