本文選題：自身免疫性葡萄膜炎 + 補體系統(tǒng)�。� 參考：《天津醫(yī)科大學(xué)》2016年博士論文

【摘要】：實驗?zāi)康?1.最近的研究結(jié)果表明,通過抑制補體系統(tǒng)的活性可以有效用于治療T細胞介導(dǎo)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,補體過敏毒素受體C3a R和C5a R對T細胞的調(diào)節(jié)作用尚不明確。因此,本課題通過比較野生型(wildtype,WT)小鼠與補體過敏毒素受體C3a R/C5a R雙基因敲除(double knockout,DKO)小鼠之間EAU的表現(xiàn),以及葡萄膜炎致病性T細胞的功能來明確C3a R和C5a R在EAU病程發(fā)展中的重要性。2.體內(nèi)的補體系統(tǒng)需要激活后才能發(fā)揮作用,雖然已有報道顯示旁路途徑的激活對EAU發(fā)病輕重產(chǎn)生影響,但是經(jīng)典途徑以及凝集素途徑的激活對EAU發(fā)病的作用還尚不清楚。C4是經(jīng)典途徑以及凝集素途徑中的共同成分,C1q是經(jīng)典途徑中的啟動因子。因此,本課題進一步通過誘導(dǎo)WT與C4KO、C1q KO小鼠的EAU,明確補體激活經(jīng)典途徑與凝集素途徑對EAU的影響。實驗方法:1.利用光感受器間維生素A類結(jié)合蛋白(interphotoreceptor binding protein,IRBP)多肽片段651-670,分別誘導(dǎo)C57BL/6J WT小鼠和C3a R/C5a R DKO小鼠EAU發(fā)病模型。免疫后,通過間接眼底鏡每日觀察疾病的發(fā)展過程,記錄下臨床評分。免疫進行兩周后,進行眼底視網(wǎng)膜成像——包括局部內(nèi)窺鏡眼底成像(topical endoscopic fundus imaging,TEFI),共焦激光掃描眼底鏡(confocal scanning laser ophthalmoscopy,c SLO),光學(xué)相干斷層掃描(spectral domain optical coherence tomography,SD-OCT)以及視網(wǎng)膜功能電生理檢查。觀察結(jié)束后,取眼球石蠟包埋切片HE染色進行組織病理學(xué)評估。主動免疫第2或3周,取脾臟分離脾細胞,在含IRBP抗原的培養(yǎng)基中進行體外培養(yǎng),另設(shè)EAU不相關(guān)抗原卵清蛋白(ovalbumin,OVA)多肽片段323-339以及無抗原組做為實驗對照,72小時后用ELISA測定細胞上清液中Th1,Th17相關(guān)細胞因子IFN-γ,IL-17。在過繼轉(zhuǎn)移誘導(dǎo)EAU發(fā)病的實驗中,分別主動免疫WT與DKO小鼠,第14天分離出脾臟中的T細胞,體外IRBP刺激培養(yǎng)擴增抗原特異性T細胞,過繼轉(zhuǎn)移至na?ve WT小鼠誘導(dǎo)眼部炎癥。2.除此之外,利用IRBP 651-670分別誘導(dǎo)WT與C4KO,C1q KO小鼠EAU模型。除上述的臨床觀察,視網(wǎng)膜成像,組織病理學(xué)評估,T細胞召回實驗以及細胞培養(yǎng)上清中IFN-γ,IL-17檢測以外,還利用IRBP 651-670特異性MHC-I/多肽四聚體復(fù)合物(Tetramer復(fù)合物)進行抗原特異性CD4陽性T細胞檢測。為比較WT與C4KO小鼠抗原提呈細胞的功能,體外分別培養(yǎng)小鼠骨髓來源樹突細胞(bone marrow derived dendritic cells,BMDCs),誘導(dǎo)成熟后與轉(zhuǎn)基因OTII小鼠的脾臟T細胞在含有OVA多肽片段323-339的條件下共培養(yǎng),72小時后利用CFSE標(biāo)記檢測脾臟細胞的增殖。為檢測WT與C4KO小鼠脾臟中致病性T細胞的活性,磁珠分選小鼠脾臟細胞中CD4陽性的T細胞,在Th0及Th1分化條件的培養(yǎng)基中體外培養(yǎng),并在不同的時間點分別使用CD25,CD69抗體檢測T細胞激活標(biāo)記物;Brdu和CFSE標(biāo)記分析T細胞增殖;Annexin V和PI染色分析T細胞凋亡;IFN-γ抗體檢測T細胞分化。實驗結(jié)果:1.EAU誘導(dǎo)后,臨床評分,視網(wǎng)膜成像,組織病理學(xué)評分的結(jié)果一致,均顯示出DKO比WT小鼠表現(xiàn)出較輕的炎癥。電生理檢測顯示DKO EAU小鼠的視網(wǎng)膜功能明顯優(yōu)于WT EAU小鼠。T細胞體外召回實驗結(jié)果表明,DKO EAU小鼠脾臟細胞對IRBP的免疫應(yīng)答低于WT對照組,表現(xiàn)為細胞培養(yǎng)上清液中IFN-γ與IL-17的水平明顯減低。另外,T細胞過繼轉(zhuǎn)移誘導(dǎo)EAU的實驗表明,來自WT EAU小鼠脾臟中的IRBP特異性T細胞,其致病能力明顯強于DKO EAU小鼠的T細胞,能夠誘導(dǎo)na?ve小鼠表現(xiàn)出更加嚴(yán)重的葡萄膜炎。2.C4KO比WT小鼠EAU的發(fā)病明顯減輕,但C1q KO與WT小鼠的炎癥表現(xiàn)無顯著差異。視網(wǎng)膜成像、組織病理學(xué)評分,以及T細胞體外召回實驗的結(jié)果與臨床觀察結(jié)果相一致。此外,Tetramer染色結(jié)果表明C4KO EAU小鼠脾臟細胞中IRBP特異性T細胞比例明顯低于WT對照組。3.C4KO小鼠骨髓來源的樹突細胞抗原提呈能力與WT小鼠并無明顯區(qū)別,但是其脾臟中CD4陽性T細胞在體外培養(yǎng)條件下活化,增殖,分化能力均不及WT小鼠CD4陽性T細胞,同時比來自WT的CD4陽性T細胞更易誘導(dǎo)凋亡。實驗結(jié)論:1.補體過敏毒素C3a和C5a及其受體C3a R和C5a R,在EAU的病程中發(fā)揮重要作用。2.補體激活經(jīng)典途徑與凝集素途徑共同成分C4對EAU的發(fā)展有顯著影響,然而經(jīng)典途徑啟動分子C1q對EAU的影響并不明顯。C4可能是通過增強T細胞的活化,增殖,分化以及抑制T細胞凋亡對EAU的發(fā)病起到促進作用。3.抑制補體系統(tǒng)的激活有可能成為治療自身免疫性葡萄膜炎的新靶點。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R773

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