细菌超抗原SEB通过角质形成细胞外泌体诱导淋巴细胞增殖的研究
发布时间：2018-08-30 16:16
【摘要】：研究背景金黄色葡萄球菌(Staphylococcus aureus)是一种常见的皮肤定植菌,它不仅可以引起多种感染性疾病,也与银屑病、湿疹、特应性皮炎等多种炎症性皮肤病关系密切。细菌超抗原引起的免疫反应是金黄色葡萄球菌致病的重要机制。角质形成细胞是表皮的主要组成细胞(约90%以上),也是细菌超抗原接触的主要细胞。已有研究表明角质形成细胞可以作为抗原提呈细胞,提呈细菌超抗原,诱导淋巴细胞增殖。然而,既往实验均使用角质形成细胞与淋巴细胞混合培养的方式来探究角质形成细胞提呈超抗原的能力,角质形成细胞是否可以通过非直接接触的方式,提呈超抗原到T细胞仍不清楚。目前一种纳米级囊泡-外泌体逐渐成为细胞间交流的研究热点。外泌体是细胞向细胞外基质中分泌的直径约30-100 nm的囊泡样小体,国内外各项研究已证实外泌体可以作为载体,携带抗原或者MHC多肽复合物到T淋巴细胞介导免疫反应。因此在本研究中我们提出假设,角质形成细胞外泌体可能作为载体,介导角质形成细胞与淋巴细胞之间信息交流,使得角质形成细胞以非接触方式提呈细菌超抗原到淋巴细胞成为可能。本研究以人角质形成细胞株(HaCa T细胞株)为研究对象,探讨HaCaT细胞是否可以在非接触的条件下辅助SEB诱导淋巴细胞增殖,并初步探究角质形成细胞是否可以通过外泌体来参与超抗原介导的免疫反应。研究方法:本研究分两部分,主要研究内容如下:第一部分:通过Transwell小室构建了HaCaT细胞与CD3+T细胞在非接触的条件下的混合培养。通过CCK8实验以及CFSE示踪法分别检测HaCaT细胞在非接触的情况下诱导T淋巴细胞增殖情况。第二部分:1.多步离心法提取HaCaT细胞外泌体,电镜检测所提取的外泌体大小及形态,Western blot检测外泌体标志蛋白CD63、Tsg101及阴性蛋白Calnexin;2.激光共聚焦显微镜检测HaCaT细胞外泌体是否可以作用于T淋巴细胞;3.Western blot检测HaCaT细胞外泌体上MHC I、MHC II、ICAM分子,探究其是否有抗原提呈的分子基础;4.CFSE示踪法检测负载SEB的HaCaT细胞分泌的外泌体是否可以诱导T淋巴细胞增殖;结果:第一部分:CCK8实验以及CFSE示踪法均证实负载SEB的HaCaT细胞可以在非接触的条件下诱导T淋巴细胞增殖;第二部分:1.多步离心法提取的HaCaT外泌体符合外泌体的基本特性,并且纯度较高;2.激光共聚焦显微镜观察到荧光标记的HaCaT外泌体可以作用于T淋巴细胞;3.HaCaT外泌体上可以检测到MHC I分子,IFN-γ处理后HaCaT外泌体上MHC I分子表达升高,且可以表达MHC II分子;4.在IFN-γ处理后,负载SEB的HaCaT细胞分泌的外泌体可以同时诱导CD4+T、CD8+T淋巴细胞的增殖。结论:许多炎症性皮肤病的发病及加重均与金黄色葡萄球菌定植关系密切,细菌超抗原引起的免疫反应是金黄色葡萄球菌致病的重要机制。我们实验证实在SEB相关的皮肤免疫中角质形成细胞以及T细胞之间存在一种非接触的作用方式,并且我们实验结果提示外泌体是角质形成细胞参与细菌超抗原相关皮肤疾病的另一途径。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R751
本文编号：2213606
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R751
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2 顾振华;汪俊军;;基质金属蛋白酶-1的研究进展[J];医学研究生学报;2006年11期
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