TIPE2在自身免疫性葡萄膜炎发病过程中的作用及机制研究
发布时间：2018-09-18 09:52
【摘要】：目的本研究的主要目的是利用IRBP1-20抗原诱导的自身免疫性葡萄膜炎疾病模型来研究TIPE2在自身免疫性葡萄膜炎发病过程中的作用,并分别从细胞水平和分子水平探讨TIPE2在此过程中的作用机制,从而为建立靶向TIPE2来控制炎症反应的新方法提供理论基础。方法1.构建EAU模型,并在野生型和TIPE2缺失C57BL/6小鼠中诱导EAU,并根据小鼠眼球病理改变来判断建模是否成功。2.野生型和TIPE2缺失C57BL/6小鼠中诱导EAU,通过比较小鼠眼球切片病理改来探讨TIPE2是否在自身免疫性葡萄膜炎病的发生和发展中起作用。3.研磨颈部引流淋巴结和I型胶原酶消化眼球组织,计数,并用IRBP、α-328刺激胞48h后ELISA方法检测炎症因子IFN-r和IL-17A的表达。4.计算并比较两组小鼠的脾脏重量和体重的比例。5.对颈部引流淋巴结、眼球组织细胞进行细胞表面染色,并用流式细胞仪检测CD4、CD8a、CD11b、CD11c及F4/80阳性的免疫细胞百分比。6.颈部淋巴结T细胞的活化标记CD62L的表达情况从而确定TIPE2抑制自身免疫性葡萄膜炎发生和发展的细胞机制。7.通过利用细胞内染色技术比较野生型小鼠和TIPE2缺失小鼠T细胞磷酸化IκBα的表达,NF-κB抑制剂Bay处理α-328刺激的T细胞,检测炎症因子IL-17A的表达,从而确定TIPE2抑制自身免疫性葡萄膜炎发生和发展的分子机制。8.研磨颈部引流淋巴结,得到淋巴结细胞悬液,用α-CD328刺激细胞后RT-PCR方法检测TIPE2 m RNA的表达情况。结果1.通过IRBP(IRBP1-20简称)抗原皮下注射可以诱导出自身免疫性葡萄膜炎疾病的病理变化,该实验结果也意味着我们在背景为C57BL/6小鼠中自身免疫性葡萄膜炎病建模成功。2.利用IRBP抗原皮下注射诱导的小鼠EAU模型,行眼球石蜡包埋病理切片并HE染色后,结果显示TIPE2缺失的小鼠HE炎症评分更高,患EAU的发病程度较野生型更严重(P0.05)。该实验结果说明TIPE2能下调自身免疫性葡萄膜炎病的发生和发展。3.通过ELISA比较野生型和TIPE2缺失小鼠在诱导自身免疫性葡萄膜炎后T细胞中炎症因子的产生,发现TIPE2缺失小鼠在颈部引流淋巴结、眼球T细胞及IRBP特异性T细胞产生的炎症因子IL-17A更多,差异有统计学意义(P0.05),IRBP特异性T细胞产生的炎症因子IFN-r同样增多,差异有统计学意义(P0.05),但是两组表达的α-CD(3+28)刺激后的IFN-r差异无统计学意义(P0.05)。4.通过比较野生型和TIPE2缺失小鼠的脾脏重量与体重比,发现两者差异无统计学意义(P0.05)。5.利用流式细胞分类技术比较野生型小鼠和TIPE2缺失小鼠在诱导自身免疫性葡萄膜炎病后淋巴结和眼球中免疫细胞(CD4、CD8a、CD11b、CD11c、F4/80)的比例,结果显示TIPE2缺失小鼠在颈部引流淋巴结及眼球处的免疫细胞(CD4、CD8a、CD11b、CD11c)差异无统计学意义(P0.05)。TIPE2缺失小鼠在眼球处的F4/80+巨噬细胞却比野生型增多,差异有统计学意义(P0.05)。6.利用流式细胞分类技术比较野生型小鼠和TIPE2缺失小鼠在发病后CD4阳性T细胞的活化,结果显示TIPE2缺失的小鼠T细胞CD62L表达较野生型降低(P0.05)。7.利用细胞内染色和流式细胞分类技术比较野生型小鼠和TIPE2缺失小鼠T细胞中IκBα的磷酸化水平,结果显示TIPE2缺失小鼠T细胞的IκBα磷酸化水平显著增加(P0.05)。NF-κB抑制剂Bay处理α-328刺激的T细胞后,炎症因子IL-17A的表达无明显差异(P0.05)。8.通过RT-PCR方法检测α-CD328刺激T细胞后TIPE2 m RNA的表达情况,结果显示TIPE2缺失小鼠T细胞的TIPE2 m RNA水平显著降低(P0.05)。结论TIPE2通过抑制IκBα的磷酸化从而抑制T细胞的活化来下调眼球中炎症因子的产生,最终抑制自身免疫性葡萄膜炎的发生和发展。
[Abstract]:Objective To study the role of TIPE2 in the pathogenesis of autoimmune uveitis by using IRBP1-20 antigen-induced autoimmune uveitis model, and to explore the mechanism of TIPE2 in the pathogenesis of autoimmune uveitis at cellular and molecular levels, so as to establish a targeted TIPE2 to control inflammatory response. Methods 1. Establish EAU model and induce EAU in wild-type and TIPE2-deficient C57BL/6 mice, and judge whether the model is successful or not according to the pathological changes of mouse eyeball. 2. Induce EAU in wild-type and TIPE2-deficient C57BL/6 mice, and investigate whether TIPE2 is autoimmune by comparing the pathological changes of mouse eyeball slices. Eyeball tissues were digested by cervical drainage lymph nodes and collagenase I and counted. The expression of inflammatory factors IFN-r and IL-17A was detected by ELISA 48 hours after IRBP and alpha-328 stimulation. 4. The ratio of spleen weight and body weight of the two groups of mice was calculated and compared. 5. The percentage of immunocytes positive for CD4, CD8a, CD11b, CD11c and F4/80 was detected by flow cytometry. 6. The expression of CD62L, an activated marker of T cells in cervical lymph nodes, was used to determine the cellular mechanism of TIPE2 inhibiting the occurrence and development of autoimmune uveitis. To compare the expression of phosphorylated I-kappa B-alpha in T cells of wild type mice and TIPE2-deficient mice. NF-kappa B inhibitor Bay treated T cells stimulated by alpha-328 and detected the expression of inflammatory factor IL-17A. The molecular mechanism of TIPE2 inhibiting the occurrence and development of autoimmune uveitis was determined. Results 1. The pathological changes of autoimmune uveitis were induced by subcutaneous injection of IRBP (IRBP1-20) antigen. The results also indicated that we successfully established the autoimmune uveitis model in C57BL/6 mice. After paraffin embedded pathological sections and HE staining, the results showed that the HE inflammation score of mice with TIPE2 deficiency was higher, and the incidence of EAU was more serious than that of wild type (P 0.05). The results showed that TIPE2 could down-regulate the occurrence and development of autoimmune uveitis. The production of inflammatory cytokines in T cells of wild-type and TIPE2-deficient mice after inducing autoimmune uveitis was higher than that of TIPE2-deficient mice in cervical drainage lymph nodes, eyeball T cells and IRBP-specific T cells (P 0.05). There was no significant difference in the expression of IFN-r between the two groups (P 0.05). 4. By comparing the spleen weight and body weight of wild type mice and TIPE2 deficient mice, it was found that there was no significant difference between them (P 0.05). 5. Flow cytometry was used to compare wild type mice and TIPE2 deficient mice. The proportion of immune cells (CD4, CD8a, CD11b, CD11c, F4/80) in lymph nodes and eyeballs of mice with TIPE2 deficiency after inducing autoimmune uveitis showed no significant difference in the number of immune cells (CD4, CD8a, CD11b, CD11c) in cervical drainage lymph nodes and eyeballs of mice with TIPE2 deficiency (P 0.05). The activation of CD4 positive T cells in wild type mice and TIPE2 deficient mice was compared by flow cytometry. The results showed that the expression of CD62L in T cells of TIPE2 deficient mice was lower than that of wild type mice (P 0.05). The phosphorylation level of I-kappa B-alpha in T cells of wild-type mice and TIPE2-deficient mice was compared. The results showed that the phosphorylation level of I-kappa B-alpha in T cells of TIPE2-deficient mice increased significantly (P 0.05). There was no significant difference in the expression of inflammatory factor IL-17A in T cells stimulated by alpha-328 after treatment with NF-kappa B inhibitor Bay (P 0.05). The expression of TIPE2 m RNA after stimulation of T cells showed that the level of TIPE2 m RNA in T cells of TIPE2-deficient mice was significantly decreased (P 0.05). Conclusion TIPE2 can inhibit the production of inflammatory factors in the eyeball by inhibiting the phosphorylation of I-kappa B-alpha and the activation of T cells, and ultimately inhibit the occurrence and development of autoimmune uveitis.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R773
本文编号：2247534
[Abstract]:Objective To study the role of TIPE2 in the pathogenesis of autoimmune uveitis by using IRBP1-20 antigen-induced autoimmune uveitis model, and to explore the mechanism of TIPE2 in the pathogenesis of autoimmune uveitis at cellular and molecular levels, so as to establish a targeted TIPE2 to control inflammatory response. Methods 1. Establish EAU model and induce EAU in wild-type and TIPE2-deficient C57BL/6 mice, and judge whether the model is successful or not according to the pathological changes of mouse eyeball. 2. Induce EAU in wild-type and TIPE2-deficient C57BL/6 mice, and investigate whether TIPE2 is autoimmune by comparing the pathological changes of mouse eyeball slices. Eyeball tissues were digested by cervical drainage lymph nodes and collagenase I and counted. The expression of inflammatory factors IFN-r and IL-17A was detected by ELISA 48 hours after IRBP and alpha-328 stimulation. 4. The ratio of spleen weight and body weight of the two groups of mice was calculated and compared. 5. The percentage of immunocytes positive for CD4, CD8a, CD11b, CD11c and F4/80 was detected by flow cytometry. 6. The expression of CD62L, an activated marker of T cells in cervical lymph nodes, was used to determine the cellular mechanism of TIPE2 inhibiting the occurrence and development of autoimmune uveitis. To compare the expression of phosphorylated I-kappa B-alpha in T cells of wild type mice and TIPE2-deficient mice. NF-kappa B inhibitor Bay treated T cells stimulated by alpha-328 and detected the expression of inflammatory factor IL-17A. The molecular mechanism of TIPE2 inhibiting the occurrence and development of autoimmune uveitis was determined. Results 1. The pathological changes of autoimmune uveitis were induced by subcutaneous injection of IRBP (IRBP1-20) antigen. The results also indicated that we successfully established the autoimmune uveitis model in C57BL/6 mice. After paraffin embedded pathological sections and HE staining, the results showed that the HE inflammation score of mice with TIPE2 deficiency was higher, and the incidence of EAU was more serious than that of wild type (P 0.05). The results showed that TIPE2 could down-regulate the occurrence and development of autoimmune uveitis. The production of inflammatory cytokines in T cells of wild-type and TIPE2-deficient mice after inducing autoimmune uveitis was higher than that of TIPE2-deficient mice in cervical drainage lymph nodes, eyeball T cells and IRBP-specific T cells (P 0.05). There was no significant difference in the expression of IFN-r between the two groups (P 0.05). 4. By comparing the spleen weight and body weight of wild type mice and TIPE2 deficient mice, it was found that there was no significant difference between them (P 0.05). 5. Flow cytometry was used to compare wild type mice and TIPE2 deficient mice. The proportion of immune cells (CD4, CD8a, CD11b, CD11c, F4/80) in lymph nodes and eyeballs of mice with TIPE2 deficiency after inducing autoimmune uveitis showed no significant difference in the number of immune cells (CD4, CD8a, CD11b, CD11c) in cervical drainage lymph nodes and eyeballs of mice with TIPE2 deficiency (P 0.05). The activation of CD4 positive T cells in wild type mice and TIPE2 deficient mice was compared by flow cytometry. The results showed that the expression of CD62L in T cells of TIPE2 deficient mice was lower than that of wild type mice (P 0.05). The phosphorylation level of I-kappa B-alpha in T cells of wild-type mice and TIPE2-deficient mice was compared. The results showed that the phosphorylation level of I-kappa B-alpha in T cells of TIPE2-deficient mice increased significantly (P 0.05). There was no significant difference in the expression of inflammatory factor IL-17A in T cells stimulated by alpha-328 after treatment with NF-kappa B inhibitor Bay (P 0.05). The expression of TIPE2 m RNA after stimulation of T cells showed that the level of TIPE2 m RNA in T cells of TIPE2-deficient mice was significantly decreased (P 0.05). Conclusion TIPE2 can inhibit the production of inflammatory factors in the eyeball by inhibiting the phosphorylation of I-kappa B-alpha and the activation of T cells, and ultimately inhibit the occurrence and development of autoimmune uveitis.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R773
【参考文献】
相关期刊论文 前9条
1 刘芮伶;阮庆国;;免疫负调控因子TIPE2在自身免疫性疾病发病过程中的作用及机制研究进展[J];国际免疫学杂志;2016年04期
2 张莲;宋继科;郭俊国;毕宏生;;Th17/Treg细胞与自身免疫性葡萄膜炎的研究进展[J];国际眼科杂志;2015年09期
3 李志;焦治兴;齐忠权;;免疫负调控分子TIPE2的研究进展[J];现代肿瘤医学;2015年13期
4 刘岚;许勇峰;;实验性自身免疫性葡萄膜视网膜炎小鼠模型的建立及观察[J];中国实用医药;2014年08期
5 王影;李洋;毕宏生;滕达;李姣;崔彦;;实验性自身免疫性葡萄膜炎炎性因子表达的动态观察[J];中华实验眼科杂志;2013年07期
6 刘正峰;崔彦;毕宏生;;先天性与获得性免疫在葡萄膜炎发生中病理机制的研究进展[J];中华实验眼科杂志;2013年05期
7 李洋;崔彦;毕宏生;;实验性自身免疫性葡萄膜炎动物模型[J];国际眼科纵览;2011年04期
8 龚文容;陈震;邢怡桥;;辅助性T细胞17和实验性自身免疫性葡萄膜炎[J];华西医学;2011年02期
9 陈颖;杨培增;;自身免疫性葡萄膜炎发病机制的研究进展[J];实用医院临床杂志;2010年06期
，本文编号：2247534
本文链接：https://www.wllwen.com/yixuelunwen/wuguanyixuelunwen/2247534.html
最近更新
教材专著
