【摘要】：研究背景金黃色葡萄球菌(Staphylococcus aureus)是一種常見的皮膚定植菌,它不僅可以引起多種感染性疾病,也與銀屑病、濕疹、特應(yīng)性皮炎等多種炎癥性皮膚病關(guān)系密切。細(xì)菌超抗原引起的免疫反應(yīng)是金黃色葡萄球菌致病的重要機制。角質(zhì)形成細(xì)胞是表皮的主要組成細(xì)胞(約90%以上),也是細(xì)菌超抗原接觸的主要細(xì)胞。已有研究表明角質(zhì)形成細(xì)胞可以作為抗原提呈細(xì)胞,提呈細(xì)菌超抗原,誘導(dǎo)淋巴細(xì)胞增殖。然而,既往實驗均使用角質(zhì)形成細(xì)胞與淋巴細(xì)胞混合培養(yǎng)的方式來探究角質(zhì)形成細(xì)胞提呈超抗原的能力,角質(zhì)形成細(xì)胞是否可以通過非直接接觸的方式,提呈超抗原到T細(xì)胞仍不清楚。目前一種納米級囊泡-外泌體逐漸成為細(xì)胞間交流的研究熱點。外泌體是細(xì)胞向細(xì)胞外基質(zhì)中分泌的直徑約30-100 nm的囊泡樣小體,國內(nèi)外各項研究已證實外泌體可以作為載體,攜帶抗原或者M(jìn)HC多肽復(fù)合物到T淋巴細(xì)胞介導(dǎo)免疫反應(yīng)。因此在本研究中我們提出假設(shè),角質(zhì)形成細(xì)胞外泌體可能作為載體,介導(dǎo)角質(zhì)形成細(xì)胞與淋巴細(xì)胞之間信息交流,使得角質(zhì)形成細(xì)胞以非接觸方式提呈細(xì)菌超抗原到淋巴細(xì)胞成為可能。本研究以人角質(zhì)形成細(xì)胞株(HaCa T細(xì)胞株)為研究對象,探討HaCaT細(xì)胞是否可以在非接觸的條件下輔助SEB誘導(dǎo)淋巴細(xì)胞增殖,并初步探究角質(zhì)形成細(xì)胞是否可以通過外泌體來參與超抗原介導(dǎo)的免疫反應(yīng)。研究方法:本研究分兩部分,主要研究內(nèi)容如下:第一部分:通過Transwell小室構(gòu)建了HaCaT細(xì)胞與CD3+T細(xì)胞在非接觸的條件下的混合培養(yǎng)。通過CCK8實驗以及CFSE示蹤法分別檢測HaCaT細(xì)胞在非接觸的情況下誘導(dǎo)T淋巴細(xì)胞增殖情況。第二部分:1.多步離心法提取HaCaT細(xì)胞外泌體,電鏡檢測所提取的外泌體大小及形態(tài),Western blot檢測外泌體標(biāo)志蛋白CD63、Tsg101及陰性蛋白Calnexin;2.激光共聚焦顯微鏡檢測HaCaT細(xì)胞外泌體是否可以作用于T淋巴細(xì)胞;3.Western blot檢測HaCaT細(xì)胞外泌體上MHC I、MHC II、ICAM分子,探究其是否有抗原提呈的分子基礎(chǔ);4.CFSE示蹤法檢測負(fù)載SEB的HaCaT細(xì)胞分泌的外泌體是否可以誘導(dǎo)T淋巴細(xì)胞增殖;結(jié)果:第一部分:CCK8實驗以及CFSE示蹤法均證實負(fù)載SEB的HaCaT細(xì)胞可以在非接觸的條件下誘導(dǎo)T淋巴細(xì)胞增殖;第二部分:1.多步離心法提取的HaCaT外泌體符合外泌體的基本特性,并且純度較高;2.激光共聚焦顯微鏡觀察到熒光標(biāo)記的HaCaT外泌體可以作用于T淋巴細(xì)胞;3.HaCaT外泌體上可以檢測到MHC I分子,IFN-γ處理后HaCaT外泌體上MHC I分子表達(dá)升高,且可以表達(dá)MHC II分子;4.在IFN-γ處理后,負(fù)載SEB的HaCaT細(xì)胞分泌的外泌體可以同時誘導(dǎo)CD4+T、CD8+T淋巴細(xì)胞的增殖。結(jié)論:許多炎癥性皮膚病的發(fā)病及加重均與金黃色葡萄球菌定植關(guān)系密切,細(xì)菌超抗原引起的免疫反應(yīng)是金黃色葡萄球菌致病的重要機制。我們實驗證實在SEB相關(guān)的皮膚免疫中角質(zhì)形成細(xì)胞以及T細(xì)胞之間存在一種非接觸的作用方式,并且我們實驗結(jié)果提示外泌體是角質(zhì)形成細(xì)胞參與細(xì)菌超抗原相關(guān)皮膚疾病的另一途徑。
[Abstract]:BACKGROUND Staphylococcus aureus is a common skin colonization bacterium. It can not only cause a variety of infectious diseases, but also closely related to psoriasis, eczema, atopic dermatitis and other inflammatory skin diseases. Keratinocytes are the main constituent cells of the epidermis (about 90%) and are also the major cells in contact with bacterial superantigens. Previous studies have shown that keratinocytes can act as antigen presenting cells, presenting bacterial superantigens and inducing lymphocyte proliferation. However, previous experiments have used the method of co-culture of keratinocytes and lymphocytes. To investigate the ability of keratinocytes to present superantigens, it is not clear whether keratinocytes can present superantigens to T cells by non-direct contact. Nowadays, a kind of nano-vesicle-exosome has gradually become a research hotspot in intercellular communication. In this study, we hypothesized that the keratinocyte exosome may act as a carrier to mediate the information exchange between keratinocytes and lymphocytes, and make the angle. It is possible for plasmacytes to present bacterial superantigens to lymphocytes in a non-contact manner. In this study, a human keratinocyte line (HaCa T cell line) was used to investigate whether HaCaT cells can assist SEB-induced lymphocyte proliferation under non-contact conditions, and whether keratinocytes can pass through exosomes. Methods: This study consists of two parts. The main contents are as follows: In the first part, HaCaT cells were co-cultured with CD3 + T cells in a Transwell chamber under non-contact conditions, and the T cells induced by HaCaT cells in non-contact conditions were detected by CCK8 assay and CFSE tracer respectively. Lymphocyte proliferation. Part II: 1. Extraction of HaCaT exosomes by multi-step centrifugation, and detection of the size and morphology of the exosomes by electron microscopy, Western blot detection of exosome markers CD63, Tsg101 and negative protein Calnexin; 2. Laser confocal microscopy to detect whether HaCaT exosomes can act on T lymphocytes; 3. Detection of MHC I, MHC II, ICAM molecules in HaCaT exosomes by RN blot to explore whether there is a molecular basis for antigen presentation; 4. CFSE tracing method to detect whether the secretion of SEB-loaded HaCaT cells can induce T lymphocyte proliferation; Results: Part I: CCK8 experiment and CFSE tracing method confirmed that SEB-loaded HaCaT cells can be in non-HaCaT cells. T lymphocyte proliferation was induced under contact conditions; Part 2: 1. HaCaT exosomes extracted by multi-step centrifugation conformed to the basic characteristics of exosomes and were of high purity; 2. Laser confocal microscopy showed that fluorescent labeled HaCaT exosomes could act on T lymphocytes; 3. MHC I molecules could be detected in HaCaT exosomes and treated with IFN-gamma. After treatment with IFN-gamma, exosomes secreted by SEB-loaded HaCaT cells could induce the proliferation of CD4+T and CD8+T lymphocytes simultaneously. Conclusion: The pathogenesis and aggravation of many inflammatory skin diseases are closely related to Staphylococcus aureus colonization and bacterial superantigen induction. The immune response is an important pathogenic mechanism of Staphylococcus aureus. We have demonstrated that there is a non-contact interaction between keratinocytes and T cells in SEB-related skin immunity, and our results suggest that exosomes are another pathway for keratinocytes to participate in bacterial superantigen-related skin diseases. Diameter.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R751

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本文編號：2213606