【摘要】：背景:抗原的交叉遞呈是指外源性抗原進(jìn)入MHC-Ⅰ類分子處理途徑的現(xiàn)象,能夠特異性啟動(dòng)細(xì)胞毒T細(xì)胞應(yīng)答,構(gòu)成防御病毒、腫瘤和胞外病原微生物的重要一環(huán),而且還參與維持機(jī)體免疫耐受的形成,與多種自身免疫性疾病的發(fā)病機(jī)制有關(guān)。樹(shù)突狀細(xì)胞抗原交叉遞呈被認(rèn)為是機(jī)體進(jìn)行外源性抗原交叉遞呈的主要途徑。外源性抗原的交叉遞呈是一系列胞內(nèi)囊泡融合、轉(zhuǎn)運(yùn)的過(guò)程,SNARE(Soluble-N-ethylmaleimide-sensitive-factor Attachment-protein Receptor)蛋白家族作為一類與囊泡識(shí)別、錨定、融合相關(guān)的功能蛋白,廣泛參與了這一過(guò)程。目的:目前對(duì)SNARE蛋白的參與交叉遞呈的具體機(jī)制還知之甚少。本課題率先在這一領(lǐng)域進(jìn)行探索,為理解樹(shù)突狀細(xì)胞抗原交叉遞呈的作用機(jī)制提供了新的思路。同時(shí),鑒定出的SNARE蛋白將為基于樹(shù)突狀細(xì)胞交叉遞呈的免疫治療提供新的靶點(diǎn)。 方法:本課題利用siRNA技術(shù)對(duì)影響DC交叉遞呈的SNARE分子進(jìn)行大規(guī)模篩選。首先,利用特異性抗原蛋白OVA刺激APC和T細(xì)胞雜交瘤,聯(lián)合ELISA方法建立起了一套穩(wěn)定的交叉遞呈能力檢測(cè)體系。然后,收集已知的SNARE分子家族成員,并利用它們的cDNA進(jìn)行siRNA的設(shè)計(jì)。接著,利用慢病毒感染的方法將siRNA轉(zhuǎn)入真核的DC2.4細(xì)胞中。最后利用穩(wěn)定表達(dá)siRNA的DC2.4細(xì)胞和交叉遞呈能力檢測(cè)體系對(duì)潛在的能夠影響交叉遞呈的SNARE分子進(jìn)行篩選。 結(jié)果:本課題最終篩選到23個(gè)與DC細(xì)胞抗原交叉遞呈相關(guān)的SNARE分子。這些分子主要分屬于2個(gè)不同的SNARE亞家族,其中Syntaxin3、Syntaxin17和Syntaxin18對(duì)交叉遞呈的影響較大。 結(jié)論:作為一類與囊泡識(shí)別、錨定、融合相關(guān)的功能蛋白,SNARE分子確實(shí)參與并影響了DC的外源性抗原交叉遞呈。這一結(jié)果為免疫調(diào)節(jié)、病毒防治和腫瘤治療等方面提供了新的理論支持和作用靶點(diǎn)。
[Abstract]:Background: the cross-presentation of antigens refers to the phenomenon of exogenous antigens entering the MHC- class I molecular processing pathway, which can specifically initiate cytotoxic T cell response and form an important part of the defense against viruses, tumors and extracellular pathogenic microorganisms. It is also involved in the formation of immune tolerance, which is related to the pathogenesis of many autoimmune diseases. Dendritic cell antigen cross-presentation is considered to be the main way to carry out cross-presentation of exogenous antigen. The cross presentation of exogenous antigen is a series of intracellular vesicle fusion, and the translocation process of SNARE (Soluble-N-ethylmaleimide-sensitive-factor Attachment-protein Receptor) protein family as a class of vesicle recognition, anchoring, fusion related functional proteins, widely involved in this process. Objective: little is known about the mechanism of SNARE protein involved in cross-presentation. This topic is the first to explore in this field, which provides a new idea for understanding the mechanism of dendritic cell antigen cross-presentation. At the same time, the identified SNARE protein will provide a new target for immunotherapy based on dendritic cell cross-presentation. Methods: siRNA technique was used to screen large-scale SNARE molecules affecting DC cross-presentation. First, the specific antigen protein OVA was used to stimulate APC and T cell hybridoma, and a stable cross-presentation capacity detection system was established with ELISA method. Then, the known members of the SNARE molecular family are collected and their cDNA is used to design the siRNA. Then, siRNA was transferred into eukaryotic DC2.4 cells by lentivirus infection. Finally, DC2.4 cells expressing siRNA stably and cross-presenting ability detection system were used to screen potential SNARE molecules that could affect cross-presentation. Results: 23 SNARE molecules associated with DC cell antigen cross-presentation were screened. These molecules mainly belong to two different SNARE subfamilies, among which Syntaxin3,Syntaxin17 and Syntaxin18 have great influence on cross-presentation. Conclusion: as a kind of functional protein associated with vesicle recognition, anchoring and fusion, SNARE does participate in and influence the cross-presentation of exogenous antigens in DC. These results provide new theoretical support and target for immunomodulation, virus prevention and cancer therapy.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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