CD226分子与临床疾病的关系及其分子机制

发布时间：2018-03-16 15:16

本文选题：CD226　切入点：白细胞介素10　出处：《第四军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：1985年,澳大利亚学者Burns G教授以混合淋巴细胞培养(Mixed Lymphocyte Culture,MLC)中活化的淋巴细胞作为免疫原免疫小鼠制备抗活化T细胞表面抗原的单克隆抗体(monoclonal antibody,mAb),筛选得到一株可特异性识别表达于活化T细胞表面膜分子的mAb,将其命名为LeoA1。随后,发现LeoA1 mAb识别的分子亦高水平表达在血小板表面,遂将此分子命名为血小板与T细胞活化抗原1(platelet and T cell activation antigen 1,PTA1)。在2000年第七届国际人类白细胞分化抗原协作组(HLDA7)大会上,由我室提交的抗PTA1 mAb和由DNAX研究所提交的抗DNAM-1mAb获准了新的CD编号-CD226,这是首次以我国实验室为主要研究单位获得的新CD分子命名。CD226分子属于免疫球蛋白超家族成员,胞膜外区含有2个V样结构域,分子量约为65k D,主要表达于T细胞、NK细胞、NKT细胞、巨核/血小板谱系、血管内皮细胞、B细胞亚群和部分造血干细胞。2003年,人CD226分子的两种配体鉴定成功,分别是人脊髓灰质炎病毒受体(PVR/CD155)和CD112。CD226分子与其配体的相互作用参与T细胞的分化和活化、NK细胞对肿瘤细胞和病毒感染细胞的杀伤、单核/巨噬细胞与内皮细胞的粘附、巨噬细胞的分化和血小板的活化与聚集等,与自身免疫性疾病、移植物抗宿主反应以及病毒感染等疾病关系密切。我们前期实验发现,CD226 mAb LeoA1能够调控人MLC培养上清中细胞因子的表达。由于MLC体系是一个多细胞培养体系,而IL-10是一个可以由多种免疫细胞合成和分泌的抑制性细胞因子(如参与固有免疫应答的树突状细胞、巨噬细胞、肥大细胞和NK细胞;参与适应性免疫应答的Th1、Th2、Th17、Treg、CD8+T细胞和B细胞),因此搞清CD226分子影响MLC体系中IL-10分泌的细胞机制十分必要;另一方面,作为具有重要免疫调节功能的细胞因子,IL-10在机体免疫应答的各阶段以及多个部位调节机体的免疫应答强度,进而参与多种疾病的发生和发展,因此进一步观测CD226分子和IL-10在临床疾病中的作用及其机制有助于深入理解CD226分子的功能。鉴于此,本课题围绕CD226分子展开研究,获得了以下的研究结果:(1)在本实验室前期发现CD226 mAb LeoA1可以调控MLC体系中细胞因子分泌格局的基础上,我们观测了LeoA1在MLC体系中上调IL-10表达的细胞机制,发现LeoA1作用于MLC体系24 h时,CD4+IL-10+T细胞的比例与对照组相比显著升高,而CD14+IL-10+细胞、CD19+IL-10+细胞、CD56+CD16+IL-10+细胞、CD11c+HLA-DR+IL-10+细胞与对照组相比无显著性差异,提示LeoA1促进CD4+IL-10+T细胞的分化;进一步分别去除PBMC中的单核细胞、CD4+T细胞或B细胞亚群,发现分别去除单核细胞和CD4+T细胞后,MLC体系中IL-10的表达水平显著降低,提示单核细胞和CD4+T细胞参与LeoA1促进MLC体系中IL-10的分泌。(2)在应用WT小鼠和Cd226-/-小鼠成功建立小鼠EAE模型的基础上,我们发现Cd226-/-小鼠EAE发病时间比WT小鼠延迟且EAE严重程度减弱;腰髓组织HE染色结果显示,Cd226-/-小鼠在EAE发病高峰期时腰髓组织中炎性细胞的浸润减少;ELISA检测血清中细胞因子水平时,发现Cd226-/-小鼠在EAE发病高峰期体内血清中IL-10表达水平较WT小鼠高。(3)在收集了120名肺癌患者和83名健康志愿者的外周血标本基础上,我们通过提取全基因组DNA并运用PCR-RFLP方法结合统计分析研究人群中CD226单核苷酸多态性位点Gly307Ser(rs763361)与肺癌易感性的关联。结果发现,在非吸烟组人群中,肺癌人群中TT基因型的比例显著高于健康对照组,OR值为3.4846,95%CI为1.1932-10.1762,P值为0.0224。女性肺癌人群中TT基因型的比例(35.14%)与健康人群(14.29%)相比无显著性差异,但P值为0.0610,提示CD226 rs763361 TT基因型可能与女性人群肺癌的易感性相关。
[Abstract]:In 1985, Professor G to Australia scholar Burns mixed lymphocyte culture (Mixed Lymphocyte Culture, MLC) monoclonal antibody in activated lymphocytes immunized mice to prepare anti activated T cell surface antigen (monoclonal, antibody, mAb), we screened a strain identified specifically expressed on activated T cell surface molecule mAb, will it was named LeoA1. LeoA1 mAb then showed the expression of molecular recognition is the high level in the platelet surface, then this molecule named platelet and T cell activation antigen 1 (platelet and T cell activation antigen 1, PTA1). In 2000 Seventh International Conference on human leukocyte differentiation antigens (HLDA7) meeting, submitted by my room mAb and anti PTA1 by DNAX research filed against DNAM-1mAb approved CD number -CD226, which is a new CD for the first time in our laboratory as the main research units to obtain the The son named.CD226 molecules belonging to the immunoglobulin superfamily, extracellular domain contains 2 V like domains, a molecular weight of about 65K D, mainly expressed in T cells, NK cells, NKT cells, megakaryocyte / platelet lineage, vascular endothelial cells, B cell subsets and hematopoietic stem cell.2003. Human CD226 molecule two ligands were successfully identified, human poliovirus receptor (PVR/CD155) differentiation and activation and interaction of CD112.CD226 and its ligands in T cells, NK cells to kill tumor cells and virus-infected cells, adhesion of monocytes / macrophages and endothelial cells, macrophage differentiation and platelet activation and aggregation, and autoimmune diseases, the relationship between graft-versus-host reaction and virus infection closely. Our previous study show, CD226 mAb LeoA1 can regulate MLC cytokines in culture supernatants of the table Da. Because the MLC system is a multi culture system, and IL-10 is a can be synthesized by a variety of immune cells and the secretion of inhibitory cytokines (such as participation in the innate immune response of dendritic cells, macrophages, mast cells and NK cells; participate in adaptive immune responses of Th1, Th2, Th17, Treg, and CD8+T cells therefore, it is necessary to B cells) cells in understanding the mechanism of CD226 molecular effects of IL-10 in MLC system secretion; on the other hand, as a important cytokine immune function and immune response intensity of IL-10 in various stages of the immune response and a plurality of parts to regulate the body's occurrence and development and participate in a variety of diseases, and its role the mechanism therefore further observations of the CD226 and IL-10 molecules in clinical diseases are helpful to understand the function of CD226 protein. In view of this, this paper focuses on the research of the CD226 molecule, were obtained using the The research results: (1) in the previous CD226 mAb LeoA1 based MLC system in the regulation of cell factor secretion pattern on the cellular mechanisms we have observed the LeoA1 expression of IL-10 in the MLC system, found that the effect of LeoA1 on the MLC system of 24 h, the percentage of CD4+IL-10+T cells was significantly higher than that in control group however, CD14+IL-10+ cells, CD19+IL-10+ cells, CD56+CD16+IL-10+ cells, CD11c+HLA-DR+IL-10+ cells showed no significant difference compared with the control group, suggesting that LeoA1 promote the differentiation of CD4+IL-10+T cells; mononuclear cells were removed further in PBMC, CD4+T cells or B cells were found, removal of mononuclear cells and CD4+T cells, the expression level of IL-10 MLC system the tip was significantly reduced, mononuclear cells and CD4+T cells in LeoA1 promoted the secretion of IL-10 in MLC system. (2) in the application of WT mice and Cd226-/ mice successfully established small Based on the model of EAE rats, we found that the onset of Cd226-/- EAE mice than in WT mice was delayed and the severity of EAE decreased; spinal cord tissue HE staining showed that inflammatory cells in Cd226-/- mice EAE the peak incidence of spinal cord tissue infiltration decreased; cytokine levels of ELISA in serum, Cd226-/- mice IL-10 in EAE the peak incidence in serum expression levels higher than those of WT mice. (3) collected in 120 lung cancer patients and 83 healthy volunteers based on peripheral blood samples, we through the extraction of genomic DNA and using PCR-RFLP method combined with statistical analysis in the study population CD226 nucleotide polymorphisms (rs763361) associated with Gly307Ser susceptibility to lung cancer. The results showed that in the non - smoking group, TT genotype in lung cancer population was significantly higher than the healthy control group, OR = 3.4846,95%CI = 1.1932-10.1762, P The proportion of TT genotype in 0.0224. female lung cancer (35.14%) was not significantly different from that in healthy population (14.29%), but P value was 0.0610, suggesting that CD226 rs763361 TT genotype may be associated with susceptibility to lung cancer in female population.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R392

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