补体系统在实验性自身免疫性葡萄膜炎中对T细胞的调节作用

发布时间：2018-07-08 20:55

本文选题：自身免疫性葡萄膜炎 + 补体系统　；参考：《天津医科大学》2016年博士论文

【摘要】：实验目的:1.最近的研究结果表明,通过抑制补体系统的活性可以有效用于治疗T细胞介导的自身免疫性疾病。然而,在自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)中,补体过敏毒素受体C3a R和C5a R对T细胞的调节作用尚不明确。因此,本课题通过比较野生型(wildtype,WT)小鼠与补体过敏毒素受体C3a R/C5a R双基因敲除(double knockout,DKO)小鼠之间EAU的表现,以及葡萄膜炎致病性T细胞的功能来明确C3a R和C5a R在EAU病程发展中的重要性。2.体内的补体系统需要激活后才能发挥作用,虽然已有报道显示旁路途径的激活对EAU发病轻重产生影响,但是经典途径以及凝集素途径的激活对EAU发病的作用还尚不清楚。C4是经典途径以及凝集素途径中的共同成分,C1q是经典途径中的启动因子。因此,本课题进一步通过诱导WT与C4KO、C1q KO小鼠的EAU,明确补体激活经典途径与凝集素途径对EAU的影响。实验方法:1.利用光感受器间维生素A类结合蛋白(interphotoreceptor binding protein,IRBP)多肽片段651-670,分别诱导C57BL/6J WT小鼠和C3a R/C5a R DKO小鼠EAU发病模型。免疫后,通过间接眼底镜每日观察疾病的发展过程,记录下临床评分。免疫进行两周后,进行眼底视网膜成像——包括局部内窥镜眼底成像(topical endoscopic fundus imaging,TEFI),共焦激光扫描眼底镜(confocal scanning laser ophthalmoscopy,c SLO),光学相干断层扫描(spectral domain optical coherence tomography,SD-OCT)以及视网膜功能电生理检查。观察结束后,取眼球石蜡包埋切片HE染色进行组织病理学评估。主动免疫第2或3周,取脾脏分离脾细胞,在含IRBP抗原的培养基中进行体外培养,另设EAU不相关抗原卵清蛋白(ovalbumin,OVA)多肽片段323-339以及无抗原组做为实验对照,72小时后用ELISA测定细胞上清液中Th1,Th17相关细胞因子IFN-γ,IL-17。在过继转移诱导EAU发病的实验中,分别主动免疫WT与DKO小鼠,第14天分离出脾脏中的T细胞,体外IRBP刺激培养扩增抗原特异性T细胞,过继转移至na?ve WT小鼠诱导眼部炎症。2.除此之外,利用IRBP 651-670分别诱导WT与C4KO,C1q KO小鼠EAU模型。除上述的临床观察,视网膜成像,组织病理学评估,T细胞召回实验以及细胞培养上清中IFN-γ,IL-17检测以外,还利用IRBP 651-670特异性MHC-I/多肽四聚体复合物(Tetramer复合物)进行抗原特异性CD4阳性T细胞检测。为比较WT与C4KO小鼠抗原提呈细胞的功能,体外分别培养小鼠骨髓来源树突细胞(bone marrow derived dendritic cells,BMDCs),诱导成熟后与转基因OTII小鼠的脾脏T细胞在含有OVA多肽片段323-339的条件下共培养,72小时后利用CFSE标记检测脾脏细胞的增殖。为检测WT与C4KO小鼠脾脏中致病性T细胞的活性,磁珠分选小鼠脾脏细胞中CD4阳性的T细胞,在Th0及Th1分化条件的培养基中体外培养,并在不同的时间点分别使用CD25,CD69抗体检测T细胞激活标记物;Brdu和CFSE标记分析T细胞增殖;Annexin V和PI染色分析T细胞凋亡;IFN-γ抗体检测T细胞分化。实验结果:1.EAU诱导后,临床评分,视网膜成像,组织病理学评分的结果一致,均显示出DKO比WT小鼠表现出较轻的炎症。电生理检测显示DKO EAU小鼠的视网膜功能明显优于WT EAU小鼠。T细胞体外召回实验结果表明,DKO EAU小鼠脾脏细胞对IRBP的免疫应答低于WT对照组,表现为细胞培养上清液中IFN-γ与IL-17的水平明显减低。另外,T细胞过继转移诱导EAU的实验表明,来自WT EAU小鼠脾脏中的IRBP特异性T细胞,其致病能力明显强于DKO EAU小鼠的T细胞,能够诱导na?ve小鼠表现出更加严重的葡萄膜炎。2.C4KO比WT小鼠EAU的发病明显减轻,但C1q KO与WT小鼠的炎症表现无显著差异。视网膜成像、组织病理学评分,以及T细胞体外召回实验的结果与临床观察结果相一致。此外,Tetramer染色结果表明C4KO EAU小鼠脾脏细胞中IRBP特异性T细胞比例明显低于WT对照组。3.C4KO小鼠骨髓来源的树突细胞抗原提呈能力与WT小鼠并无明显区别,但是其脾脏中CD4阳性T细胞在体外培养条件下活化,增殖,分化能力均不及WT小鼠CD4阳性T细胞,同时比来自WT的CD4阳性T细胞更易诱导凋亡。实验结论:1.补体过敏毒素C3a和C5a及其受体C3a R和C5a R,在EAU的病程中发挥重要作用。2.补体激活经典途径与凝集素途径共同成分C4对EAU的发展有显著影响,然而经典途径启动分子C1q对EAU的影响并不明显。C4可能是通过增强T细胞的活化,增殖,分化以及抑制T细胞凋亡对EAU的发病起到促进作用。3.抑制补体系统的激活有可能成为治疗自身免疫性葡萄膜炎的新靶点。
[Abstract]:Objective: 1. recent results suggest that the activity of the complement system can be effectively used to treat T cells mediated autoimmune diseases. However, in autoimmune uveitis (experimental autoimmune uveitis, EAU), the regulatory role of complement allergic toxin receptor C3a R and C5a R to T cells is not clear. In this study, we compare the expression of EAU between the wildtype (WT) mice and the complement allergic toxin receptor C3a R/C5a R double gene knockout (double knockout, DKO) mice, as well as the function of the pathogenicity T cells of the uveitis to define the C3a R and the vital complement system in the development of the disease course. Although it has been reported that activation of the pathway of the bypass has an effect on the severity of EAU, the role of the classical pathway and the activation of the agglutinin pathway for the pathogenesis of EAU remains unclear..C4 is a common component in the classical pathway and in the lectin pathway. C1q is the starting factor in the classical pathway. Therefore, the subject is further induced by induction. WT and the EAU of C4KO, C1q KO mice, clarify the effect of complement activating classical pathways and agglutinin pathway on EAU. Experimental methods: 1. using the vitamin A binding protein (interphotoreceptor binding protein, IRBP) polypeptide fragment between the photoreceptor (interphotoreceptor binding protein, IRBP) 651-670, respectively. The development process of the disease was observed daily by indirect ophthalmoscope, and the clinical score was recorded. After two weeks of immunization, fundus retina imaging - including topical endoscopic fundus imaging (TEFI), confocal laser scanning ophthalmoscope (confocal scanning laser ophthalmoscopy, C SLO), optical coherence tomography Spectral domain optical coherence tomography (SD-OCT) and retinal function electrophysiological examination. After the observation, the paraffin embedded section of the eyeball was examined by HE staining for histopathology. The spleen cells were isolated from the spleen for second or 3 weeks, and were cultured in the culture medium containing IRBP antigen, and the ovum of EAU unrelated antigen was set up. Ovalbumin (OVA) polypeptide fragment 323-339 and no antigen group were used as experimental control. After 72 hours, ELISA was used to determine Th1, Th17 related cytokine IFN- gamma in cell supernatant. IL-17. was actively immunized with WT and DKO mice in the experiment of adoptive metastasis induced EAU, and T cells in the spleen were isolated in fourteenth days. In vitro IRBP stimulates culture. Amplification of antigen specific T cells, adoptive transfer to na? Ve WT mice induced eye inflammation.2., using IRBP 651-670 to induce WT and C4KO, C1q KO mice EAU model. Besides the above clinical observation, retinal imaging, histopathology evaluation, T cell recall experiment and detection of cell culture supernatant BP 651-670 specific MHC-I/ polypeptide four polymer complex (Tetramer complex) was used for the detection of antigen specific CD4 positive T cells. In order to compare the function of antigen presenting cells in WT and C4KO mice, the mouse bone marrow derived dendritic cells (bone marrow derived dendritic cells) were cultured in vitro, and the spleen was induced and the spleen of transgenic mice after maturation was induced. The dirty T cells were co cultured under the condition of OVA polypeptide fragment 323-339. After 72 hours, the proliferation of spleen cells was detected by CFSE markers. In order to detect the activity of pathogenic T cells in the spleen of WT and C4KO mice, the CD4 positive T cells in the spleen cells of the mice were cultured in vitro, and were cultured in vitro in the medium of Th0 and Th1 differentiation conditions. CD25, CD69 antibodies were used to detect T cell activation markers, Brdu and CFSE markers were used to analyze T cell proliferation; T cell apoptosis was analyzed by Annexin V and PI staining; IFN- gamma antibody was used to detect the differentiation of T cells. Electrophysiological tests showed that the retinal function of DKO EAU mice was obviously better than that of.T cells in WT EAU mice in vitro. The results showed that the immune response of spleen cells to IRBP in DKO EAU mice was lower than that of WT control group, and that the level of IFN- gamma and IL-17 in cell culture supernatant was significantly reduced. Moreover, T cells were adoptive and transferred. The experiment of inducing EAU showed that the IRBP specific T cells from the spleen of WT EAU mice were significantly stronger than T cells in DKO EAU mice, and could induce na? Ve mice to show more severe uveitis than those in WT mice. In addition, the results of Tetramer staining showed that the proportion of IRBP specific T cells in the spleen cells of C4KO EAU mice was significantly lower than that of the WT control group, and that the antigen presenting ability of the dendritic cells from the bone marrow derived from the.3.C4KO mice of the WT control group was not significantly different from that of the WT mice, but the results showed that there was no significant difference between the C4KO EAU mice and the WT mice. The CD4 positive T cells in the spleen were activated in vitro, and the proliferation and differentiation were less than that of CD4 positive T cells in WT mice, while the CD4 positive T cells from WT were more likely to induce apoptosis. C4, a common component of lectin pathway, has a significant effect on the development of EAU. However, the effect of the classical pathway promoter C1q on EAU is not obvious.C4 may be the promotion of the activation, proliferation, differentiation and inhibition of T cell apoptosis to the pathogenesis of EAU, and the activation of the.3. suppressing complement system is likely to be the treatment of autoimmune disease. A new target for uveitis.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R773

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