TIPE2在自身免疫性葡萄膜炎發(fā)病過(guò)程中的作用及機(jī)制研究
發(fā)布時(shí)間：2018-09-18 09:52
【摘要】：目的本研究的主要目的是利用IRBP1-20抗原誘導(dǎo)的自身免疫性葡萄膜炎疾病模型來(lái)研究TIPE2在自身免疫性葡萄膜炎發(fā)病過(guò)程中的作用,并分別從細(xì)胞水平和分子水平探討TIPE2在此過(guò)程中的作用機(jī)制,從而為建立靶向TIPE2來(lái)控制炎癥反應(yīng)的新方法提供理論基礎(chǔ)。方法1.構(gòu)建EAU模型,并在野生型和TIPE2缺失C57BL/6小鼠中誘導(dǎo)EAU,并根據(jù)小鼠眼球病理改變來(lái)判斷建模是否成功。2.野生型和TIPE2缺失C57BL/6小鼠中誘導(dǎo)EAU,通過(guò)比較小鼠眼球切片病理改來(lái)探討TIPE2是否在自身免疫性葡萄膜炎病的發(fā)生和發(fā)展中起作用。3.研磨頸部引流淋巴結(jié)和I型膠原酶消化眼球組織,計(jì)數(shù),并用IRBP、α-328刺激胞48h后ELISA方法檢測(cè)炎癥因子IFN-r和IL-17A的表達(dá)。4.計(jì)算并比較兩組小鼠的脾臟重量和體重的比例。5.對(duì)頸部引流淋巴結(jié)、眼球組織細(xì)胞進(jìn)行細(xì)胞表面染色,并用流式細(xì)胞儀檢測(cè)CD4、CD8a、CD11b、CD11c及F4/80陽(yáng)性的免疫細(xì)胞百分比。6.頸部淋巴結(jié)T細(xì)胞的活化標(biāo)記CD62L的表達(dá)情況從而確定TIPE2抑制自身免疫性葡萄膜炎發(fā)生和發(fā)展的細(xì)胞機(jī)制。7.通過(guò)利用細(xì)胞內(nèi)染色技術(shù)比較野生型小鼠和TIPE2缺失小鼠T細(xì)胞磷酸化IκBα的表達(dá),NF-κB抑制劑Bay處理α-328刺激的T細(xì)胞,檢測(cè)炎癥因子IL-17A的表達(dá),從而確定TIPE2抑制自身免疫性葡萄膜炎發(fā)生和發(fā)展的分子機(jī)制。8.研磨頸部引流淋巴結(jié),得到淋巴結(jié)細(xì)胞懸液,用α-CD328刺激細(xì)胞后RT-PCR方法檢測(cè)TIPE2 m RNA的表達(dá)情況。結(jié)果1.通過(guò)IRBP(IRBP1-20簡(jiǎn)稱(chēng))抗原皮下注射可以誘導(dǎo)出自身免疫性葡萄膜炎疾病的病理變化,該實(shí)驗(yàn)結(jié)果也意味著我們?cè)诒尘盀镃57BL/6小鼠中自身免疫性葡萄膜炎病建模成功。2.利用IRBP抗原皮下注射誘導(dǎo)的小鼠EAU模型,行眼球石蠟包埋病理切片并HE染色后,結(jié)果顯示TIPE2缺失的小鼠HE炎癥評(píng)分更高,患EAU的發(fā)病程度較野生型更嚴(yán)重(P0.05)。該實(shí)驗(yàn)結(jié)果說(shuō)明TIPE2能下調(diào)自身免疫性葡萄膜炎病的發(fā)生和發(fā)展。3.通過(guò)ELISA比較野生型和TIPE2缺失小鼠在誘導(dǎo)自身免疫性葡萄膜炎后T細(xì)胞中炎癥因子的產(chǎn)生,發(fā)現(xiàn)TIPE2缺失小鼠在頸部引流淋巴結(jié)、眼球T細(xì)胞及IRBP特異性T細(xì)胞產(chǎn)生的炎癥因子IL-17A更多,差異有統(tǒng)計(jì)學(xué)意義(P0.05),IRBP特異性T細(xì)胞產(chǎn)生的炎癥因子IFN-r同樣增多,差異有統(tǒng)計(jì)學(xué)意義(P0.05),但是兩組表達(dá)的α-CD(3+28)刺激后的IFN-r差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4.通過(guò)比較野生型和TIPE2缺失小鼠的脾臟重量與體重比,發(fā)現(xiàn)兩者差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。5.利用流式細(xì)胞分類(lèi)技術(shù)比較野生型小鼠和TIPE2缺失小鼠在誘導(dǎo)自身免疫性葡萄膜炎病后淋巴結(jié)和眼球中免疫細(xì)胞(CD4、CD8a、CD11b、CD11c、F4/80)的比例,結(jié)果顯示TIPE2缺失小鼠在頸部引流淋巴結(jié)及眼球處的免疫細(xì)胞(CD4、CD8a、CD11b、CD11c)差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。TIPE2缺失小鼠在眼球處的F4/80+巨噬細(xì)胞卻比野生型增多,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。6.利用流式細(xì)胞分類(lèi)技術(shù)比較野生型小鼠和TIPE2缺失小鼠在發(fā)病后CD4陽(yáng)性T細(xì)胞的活化,結(jié)果顯示TIPE2缺失的小鼠T細(xì)胞CD62L表達(dá)較野生型降低(P0.05)。7.利用細(xì)胞內(nèi)染色和流式細(xì)胞分類(lèi)技術(shù)比較野生型小鼠和TIPE2缺失小鼠T細(xì)胞中IκBα的磷酸化水平,結(jié)果顯示TIPE2缺失小鼠T細(xì)胞的IκBα磷酸化水平顯著增加(P0.05)。NF-κB抑制劑Bay處理α-328刺激的T細(xì)胞后,炎癥因子IL-17A的表達(dá)無(wú)明顯差異(P0.05)。8.通過(guò)RT-PCR方法檢測(cè)α-CD328刺激T細(xì)胞后TIPE2 m RNA的表達(dá)情況,結(jié)果顯示TIPE2缺失小鼠T細(xì)胞的TIPE2 m RNA水平顯著降低(P0.05)。結(jié)論TIPE2通過(guò)抑制IκBα的磷酸化從而抑制T細(xì)胞的活化來(lái)下調(diào)眼球中炎癥因子的產(chǎn)生,最終抑制自身免疫性葡萄膜炎的發(fā)生和發(fā)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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R773
本文編號(hào)：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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R773
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