本文選題：CD226　切入點：白細(xì)胞介素10　出處：《第四軍醫(yī)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：1985年,澳大利亞學(xué)者Burns G教授以混合淋巴細(xì)胞培養(yǎng)(Mixed Lymphocyte Culture,MLC)中活化的淋巴細(xì)胞作為免疫原免疫小鼠制備抗活化T細(xì)胞表面抗原的單克隆抗體(monoclonal antibody,mAb),篩選得到一株可特異性識別表達于活化T細(xì)胞表面膜分子的mAb,將其命名為LeoA1。隨后,發(fā)現(xiàn)LeoA1 mAb識別的分子亦高水平表達在血小板表面,遂將此分子命名為血小板與T細(xì)胞活化抗原1(platelet and T cell activation antigen 1,PTA1)。在2000年第七屆國際人類白細(xì)胞分化抗原協(xié)作組(HLDA7)大會上,由我室提交的抗PTA1 mAb和由DNAX研究所提交的抗DNAM-1mAb獲準(zhǔn)了新的CD編號-CD226,這是首次以我國實驗室為主要研究單位獲得的新CD分子命名。CD226分子屬于免疫球蛋白超家族成員,胞膜外區(qū)含有2個V樣結(jié)構(gòu)域,分子量約為65k D,主要表達于T細(xì)胞、NK細(xì)胞、NKT細(xì)胞、巨核/血小板譜系、血管內(nèi)皮細(xì)胞、B細(xì)胞亞群和部分造血干細(xì)胞。2003年,人CD226分子的兩種配體鑒定成功,分別是人脊髓灰質(zhì)炎病毒受體(PVR/CD155)和CD112。CD226分子與其配體的相互作用參與T細(xì)胞的分化和活化、NK細(xì)胞對腫瘤細(xì)胞和病毒感染細(xì)胞的殺傷、單核/巨噬細(xì)胞與內(nèi)皮細(xì)胞的粘附、巨噬細(xì)胞的分化和血小板的活化與聚集等,與自身免疫性疾病、移植物抗宿主反應(yīng)以及病毒感染等疾病關(guān)系密切。我們前期實驗發(fā)現(xiàn),CD226 mAb LeoA1能夠調(diào)控人MLC培養(yǎng)上清中細(xì)胞因子的表達。由于MLC體系是一個多細(xì)胞培養(yǎng)體系,而IL-10是一個可以由多種免疫細(xì)胞合成和分泌的抑制性細(xì)胞因子(如參與固有免疫應(yīng)答的樹突狀細(xì)胞、巨噬細(xì)胞、肥大細(xì)胞和NK細(xì)胞;參與適應(yīng)性免疫應(yīng)答的Th1、Th2、Th17、Treg、CD8+T細(xì)胞和B細(xì)胞),因此搞清CD226分子影響MLC體系中IL-10分泌的細(xì)胞機制十分必要;另一方面,作為具有重要免疫調(diào)節(jié)功能的細(xì)胞因子,IL-10在機體免疫應(yīng)答的各階段以及多個部位調(diào)節(jié)機體的免疫應(yīng)答強度,進而參與多種疾病的發(fā)生和發(fā)展,因此進一步觀測CD226分子和IL-10在臨床疾病中的作用及其機制有助于深入理解CD226分子的功能。鑒于此,本課題圍繞CD226分子展開研究,獲得了以下的研究結(jié)果:(1)在本實驗室前期發(fā)現(xiàn)CD226 mAb LeoA1可以調(diào)控MLC體系中細(xì)胞因子分泌格局的基礎(chǔ)上,我們觀測了LeoA1在MLC體系中上調(diào)IL-10表達的細(xì)胞機制,發(fā)現(xiàn)LeoA1作用于MLC體系24 h時,CD4+IL-10+T細(xì)胞的比例與對照組相比顯著升高,而CD14+IL-10+細(xì)胞、CD19+IL-10+細(xì)胞、CD56+CD16+IL-10+細(xì)胞、CD11c+HLA-DR+IL-10+細(xì)胞與對照組相比無顯著性差異,提示LeoA1促進CD4+IL-10+T細(xì)胞的分化;進一步分別去除PBMC中的單核細(xì)胞、CD4+T細(xì)胞或B細(xì)胞亞群,發(fā)現(xiàn)分別去除單核細(xì)胞和CD4+T細(xì)胞后,MLC體系中IL-10的表達水平顯著降低,提示單核細(xì)胞和CD4+T細(xì)胞參與LeoA1促進MLC體系中IL-10的分泌。(2)在應(yīng)用WT小鼠和Cd226-/-小鼠成功建立小鼠EAE模型的基礎(chǔ)上,我們發(fā)現(xiàn)Cd226-/-小鼠EAE發(fā)病時間比WT小鼠延遲且EAE嚴(yán)重程度減弱;腰髓組織HE染色結(jié)果顯示,Cd226-/-小鼠在EAE發(fā)病高峰期時腰髓組織中炎性細(xì)胞的浸潤減少;ELISA檢測血清中細(xì)胞因子水平時,發(fā)現(xiàn)Cd226-/-小鼠在EAE發(fā)病高峰期體內(nèi)血清中IL-10表達水平較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標(biāo)本基礎(chǔ)上,我們通過提取全基因組DNA并運用PCR-RFLP方法結(jié)合統(tǒng)計分析研究人群中CD226單核苷酸多態(tài)性位點Gly307Ser(rs763361)與肺癌易感性的關(guān)聯(lián)。結(jié)果發(fā)現(xiàn),在非吸煙組人群中,肺癌人群中TT基因型的比例顯著高于健康對照組,OR值為3.4846,95%CI為1.1932-10.1762,P值為0.0224。女性肺癌人群中TT基因型的比例(35.14%)與健康人群(14.29%)相比無顯著性差異,但P值為0.0610,提示CD226 rs763361 TT基因型可能與女性人群肺癌的易感性相關(guān)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R392

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1 曾漢玉;CD226分子與臨床疾病的關(guān)系及其分子機制[D];第四軍醫(yī)大學(xué);2016年

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