【作者】 王东琳；

【导师】 金伯泉； 杨琨；

【作者基本信息】 第四军医大学 ， 免疫学， 2008， 博士

【摘要】 人CD226曾先后被称为T细胞谱系特异性活化抗原(1 TLiSA1,1985)、血小板T细胞活化抗原1(PTA1,1989)DNAX辅助分子1(DNAM-1,1996),并于2000年首次以我国实验室为主要研究单位在国际人类白细胞分化抗原协作组(HLDA)大会上获得编号为CD226。该分子于1996年基因克隆成功,cDNA全长2603bp,包含7个外显子,开放读框编码336个氨基酸,胞浆区具有磷酸化及与信号分子相互作用的位点。CD226分子与表达于杀伤性T细胞(CTL)和NK细胞的CD96(Tactile)分子在蛋白水平有22%的同源性,与BGP-1、CD36、PRR和果蝇神经胶质蛋白Neuroglian也有一定的同源性。CD226成熟蛋白由318个氨基酸组成,分子量约为65kDa,属免疫球蛋白超家族I型跨膜糖蛋白,表达于T细胞、NK细胞、NKT细胞、单核细胞、巨核/血小板谱系及活化的血管内皮细胞;广泛参与了免疫系统的生理和病理学功能,如T细胞、巨核细胞的分化,NK细胞对肿瘤细胞、病毒感染细胞的杀伤,血小板的活化与聚集,单核/巨噬细胞穿越血管内皮细胞等过程,并参与其中的信号转导。CD226分子体内天然配体于2003年得到鉴定,为同属于nectin/Necl家族的黏附分子:人脊髓灰质炎病毒受体(PVR/Necl-5/CD155)及其家族成员人PVR受体相关分子(PRR-2/nectin-2/CD112),CD112同时也是单纯疱疹病毒(HSV-1和HSV-2)的受体。小鼠PTA1/CD226基因于2002年成功克隆,由7个外显子组成,编码完整小鼠CD226分子的cDNA开放读框有1002bp,编码333个氨基酸残基,小鼠CD226分子亦属免疫球蛋白超家族I型跨膜糖蛋白。小鼠CD226与人CD226在核苷酸水平有67%的同源性,而在氨基酸水平有53%的同源性。已证实mCD112和mCD155(Tage4)是mCD226的配体。小鼠CD226介导了抗原特异性CD8+T细胞共刺激信号的转导,并可参与肿瘤的免疫监视,而小鼠CD226分子在自身免疫调节中的作用尚未有较多的研究结果。Th17细胞和Th1细胞均可诱导自身免疫应答,但其二者的相互关系尚未清楚。EAE最初被认为是由Th1细胞介导的,但近期的越来越多的研究认为,部分器官特异性自身免疫性动物模型中,Th17细胞对于诱导整个免疫病理中是必不可少的,并提出EAE首先是由Th17细胞启动介导的理论。本实验就小鼠CD226分子在体外培养体系及体内功能实验所参与的自身免疫调节进行了初步研究。本研究中首先克隆化建立稳定表达mPTA1-Fc融合蛋白的CHO-mPTA1/Ig细胞株,通过培养上清制备mPTA1-Fc融合蛋白,并成功免疫新西兰大白兔动物,获得了较高的免疫血清,通过亲和层析的方法纯化出特异性的多抗,ELISA、SDS-PAGE电泳结果及免疫荧光流式检测显示,该多克隆抗体具有良好的生物学活性和较高的纯度。在单抗制备过程中,将融合蛋白免疫大鼠,利用杂交瘤技术,建立制备大鼠源性单抗的平台,从5400个克隆孔中,通过交叉筛选挑选出5株阳性克隆,利用裸鼠体内诱生腹水及无血清培养上清纯化获得了具有较高活性的单抗克隆抗体。所获得的5株单抗经ELISA、SDS-PAGE电泳结果及免疫荧光流式检测显示,有4株能很好的识别小鼠CD226的重组蛋白及天然分子,为后续研究mPTA1分子的表达、分布及功能提供了有利的研究工具。我们利用所制备的抗体通过免疫荧光染色技术检测小鼠PTA1在不同细胞系的表达情况。结果表明,在小鼠肥大细胞系、小鼠单核/巨噬细胞系PTA1分子表达较高。此外mPTA1分子在小鼠树突状细胞系表达较高,在B细胞也有组成性的表达。而在上皮细胞来源、纤维母细胞或低分化细胞来源细胞系上表达为阴性。本研究还发现正常小鼠脾淋巴细胞即有mPTA1分子的高表达,CD4+T细胞及CD8+T细胞均表达mPTA1分子,但CD8+T细胞表达更高,小鼠NK细胞及巨噬细胞表面也有mPTA1分子的表达。小鼠脾淋巴细胞经MLC培养7天后,其mPTA1的表达更加明显,CD8+mPTA1+细胞与CD4+mPTA1+细胞均有明显增多,而且以CD8+mPTA1+细胞更为显著。同时发现,作为小鼠PBMC的脾淋巴细胞在经MLC刺激的同时,与PTA1抗体共培养后,其细胞培养上清中Th1样细胞因子INF-γ水平明显升高,胞内细胞因子的荧光标记染色结果也有类似变化。提示PTA1分子可能在CD4+Th细胞分化中发挥了重要的免疫调节作用。为进一步研究小鼠PTA1分子所参与的免疫调节机制作用,我们又进行了mPTA1的体内功能实验。首先我们成功建立了自身免疫性疾病的经典动物模型实验性自身免疫性脑脊髓炎(EAE)。用髓鞘少突胶质细胞糖蛋白(MOG)35-55肽段并辅以结核杆菌及百日咳毒素免疫EAE敏感动物品系C57BL/6小鼠后,于免疫免疫后第10天开始陆续发病,出现EAE的典型临床神经系统症状,病程为慢性单时相进展型,约在第20天达到发病高峰,发病率达到70%,同时伴随脑组织的病理改变, HE染色检测结果发现,在脑白质区小血管周围有淋巴细胞浸润,即呈“袖套样”炎性结构改变。LFB染色发现在脑的冠状切片及脊髓纵向矢状切片中发现,白质区有LFB未着色区,髓鞘稀疏不连续,部分呈片状脱失。EAE模型小鼠的脾淋巴细胞IFN-γ的胞内染色水平表达较正常小鼠有明显下降,而IL-17水平增高了近70%。当小鼠EAE模型中注射mPTA1抗体时,发现EAE的发病率、发病时间及发病程度均有较好的缓解,发病率由60%减少到30%,发病起始时间由免疫后的第7天延缓至第14天,平均临床分数由2.2分降低至0.5分。同时,在对EAE组和抗体注射组的研究中发现,抗体注射组的脾淋巴细胞IFN-γ的胞内染色水平较EAE组升高了约85%,接近正常未免疫小鼠;而IL-17水平反而较EAE组降低了约87%。这些研究结果提示我们,抗mPTA1抗体对EAE有较强的保护机制之一可能是由于通过提高了EAE小鼠机体内IFN-γ的水平,从而抑制了IL-17的产生,进而减弱了IL-17诱导的自身免疫性炎症。更多还原

【Abstract】 Human CD226 platelet and T cell activation antigen 1 (PTA1), also named as T lineage-specific activation antigen 1 (TLiSA1,1985) and DNAX accessory molecule-1(DNAM-1,1996) was designated as CD226 in the 7th Workshop and Conference on Human Leukocyte Differentiation Antigen (HLDA7) in 2000. In 1996, Human CD226 gene was cloned. The full length of human CD226 cDNA has 2603bp, and its ORF encoding 336 amino acids is composed of 7 exons and 6 introns. There are some phosphorylation sites in its cytoplasmic region. At protein level, CD226 shows 22% homology with CD96 (Tactile) which is expressed on activated T cells and NK cells, and also shares certain homology to BGP-1, CD36, PRR, and drosophila nerve colloid protein-Neuroglian. As a member of the immunoglobulin superfamily (IgSF), CD226 is a 65kDaⅠtype transmembrane glycoprotein, and composed of 318 amino acids. Human CD226 is expressed on cell surface of T cells, NK cells, NKT cells, monocytes/macrophages, megakaryocytes and platelets, and activated vascular endothelial cells, which is involved in a variety of immunological functions, including the differentiation of T cells and megakaryocytes, the cytotoxicity of NK cells against tumor cells and virus infected cells, the activation and aggregation of platelets, and the transmigration of monocytes/macrophages through vascular endothelial cells.In 2003, human poliovirus receptor (PVR/Necl-5/CD155) and its nectin/Necl (nectin-like) family member poliovirus receptor related 2 (PRR-2/ nectin-2/CD112) were identified as the ligands for CD226. CD112 also serves as the receptor for herpes simplex virus (HSV-1 and HSV-2).The cDNA encoding murine PTA1/CD226 (mPTA1/mCD226) was cloned in 2002, which is composed of 7 exons. It was shown that the ORF of murine CD226 has 1002bp, encoding 333 amino acids. The mPTA1 isⅠtype transmembrane glycoprotein, and also belongs to the immunoglobulin superfamily. Murine PTA1 has 67% homology with human PTA1 at cDNA level, and 53% homology at amino acid level. It has been demonstrated that murine CD112 and murine CD155 (Tage 4) are the ligands for murine PTA1/CD226. Murine CD226 can mediate the costimulatory signal in antigen-specific CD8+ T cell, and also participate in cell-mediated tumor specific immunity. Nevertheless, there are not sufficient researches whether and how mPTA1 play the function of immunological regulation in the auto-immunity. Both Th17 cells and Th1 cells could induce the auto-immunity response. However the relationship of the two kinds of cells is not clear. Th1 cells were once thought to be the key induction factor in EAE. Along with more and more researchs, it recently is considered that Th17 cells are indispensable to induce whole immunopathogenesis in organ-specific autoimmunity in animal model. And the theory also has been presented that EAE is primed firstly by Th17 cells. The laboratory mouse holds a special place in biology as the most accessible mammalian genetic system and therefore the organism of first resort in modeling many human trails and disorders. Therefore, in order to explore the biological function and pathological significance, we started to investigate the effect of mCD226 on pathogeny of experimental autoimmune encephalomyelitis.At first, we cloned and stabilized the CHO-mPTA1/Ig cell strain secreting mPTA1-Fcfusion protein and prepared the mPTA1-Fc fusion protein by purifing the culture supernatant. Then we gained the immune serum after immunizing New-Zealand rabbit using mCD226-Fc fusion protein. With the affinity chromatograph technology, we purified the specific poly antibodies against mPTA1. These antibodies have the satisfactory biologic activity and higher purity detected by ELISA, SDS-PAGE electrophoresis and immunofluorescence. Meanwhile, we immunized SD rat with the fusion protein and set up the system of preparing rat monoclone antibody against murine PTA1. Five positive hybrid strains from 5400 clones were selected by ELISA screening. Specific monoclone antibodies against mPTA1 were obtained from the ascitic fluid derivated from nude mice and cell culture supernatant, and could be used for detecting both the fusion protein and the natural murine PTA1 molecule which provide a favourable tool of for research on the expression,distribution and function of murine PTA1.Using these specific antibodies against murine PTA1, the expression of mPTA1 on various murine cell lineage was investigated by immunofluorescence staining technique. The results showed that mPTA1 expressed on the surface of murine mast cell, monocyte/macrophage cell lines, murine dendritic cell and a portion of B cells, but not cells origined from epithelium, fibroblast and poor-differentiated cells.We also found that normal mice splenic cells expressed mPTA1 including T cells, NK cells and macrophages. Although CD4+ T cells and CD8+ T cells all expressed the mPTA1 molecule, and CD8+ T cells expressed higher level of mPTA1 than CD4+ T cells. The expression of mPTA1 on murine splenic lymphocytes (both CD4+ T cells and CD8+ T cells) was increased after alloatigen simulation, MLC.At the same time, we found that the level of IFN-γfrom cell culture supernants in MLC was elevated in the presence of anti-mPTA1 andibodies. The similarly results also could be gained by detecting fluorescent labeling intracellular IFN-γ.To investigate the regulatory effect of mPTA1 antibodies on autoimmune diseases in vivo, we set up the murine experimental autoimmune encephalomyelitis (EAE) model. Immunizing C57BL/6 mice with MOG35-55 peptide plus Bacillus tuberculosis and pertussis toxin, the clinical disorder in nervous system appeared on day 10 after the peptide immunization. Histological examination revealed inflammatory foci characteristic of EAE throughout the CNS of affected animals with typical perivascular infiltration of mononuclear cells. Secondary demyelination was also observed. The level of IFN-γin the EAE mice splenic lymphocyte also was decreased by intracellular cytokine staining, whereas the level of IL-17 was raised up sixty percentages.The administration of anti-mPTA1 antibody could significantly decrease the disease incidence, delay the disease onset and reduce the severity of the initial phase of EAE compared with the control group. The EAE disease incidence of the group with antibody treatment downed from 60% to 30%. The disease onset was postponed on day 14 after immunizing. The clinical mean score of treatment group was cut downed from 2.2 to 0.5. The intracellular level of IFN-γin antibody treatment mice splenic lymphocyte compared with the control group was raised up 85% whereas the level of IL-17 was decreased 87%. So we assumed that PTA1 plays an important role in autoimmunity inflammation by regulating the producing of IFN-γwhich inhibited the Th17 cells in mice.更多还原