IL-22对角质形成细胞表达HB-EGF及TIG3作用机制的研究

发布时间：2018-07-17 02:46

【摘要】：银屑病是临床常见的慢性易复发的红斑、鳞屑性疾病。本病临床可分为寻常型、关节病型、红皮病型、脓疱型4个类型。病程慢性,反复发作。其发病机理尚未完全明确。目前研究认为银屑病的发病机制主要是由辅助性Th细胞及其相关细胞因子介导的以角质形成细胞为靶点的炎症反应。Th22细胞是新发现的CD4+辅助性T细胞亚群,在银屑病外周血中高表达,其主要细胞因子为IL-22。通过其实现相关生物学功能。在银屑病病人外周血及皮损中IL-22水平和银屑病的严重程度呈明显的正相关。我们的前期研究表明肝素结合表皮生长因子样生长因子(heparin-binding epidermal-growth-factor-like growth factor,HB-EGF)在银屑病表皮KC异常增殖和其转归中发挥重要作用。本课题的前期研究表明IL-22可以诱导体外培养Ha Ca T细胞表达HB-EGF,但作用机制未明,丝裂原活化蛋白激酶(MAPK-ERK1/2)通路和转录激活因子途径(JAK/STAT)这两种信号通路在银屑病发病机制中起不可或缺作用。他扎罗汀诱导基因3(Tazarotene-induced gene 3,TIG3)是一种肿瘤抑制因子。TIG3可以调节角质形成细胞的增殖和分化,在银屑病表达降低。综上所述,我们推测IL-22促KC分泌HB-EGF可能与MAPK-ERK1/2和JAK/STAT这两条信号通路有关,且与TIG3表达异常存在一定关系。IL-22通过促进HB-EGF表达,抑制TIG3表达参与银屑病发生、发展。本实验研究IL-22促HaCaT细胞分泌HB-EGF作用机制及对HaCaT细胞分泌TIG3的影响。目的:研究白介素22(Interleukin-22,IL-22)对角质形成细胞表达HB-EGF的作用机制和对TIG3表达影响及作用机制。方法:1、以不同浓度(12.5μg/L、25μg/L、50μg/L、100μg/L)IL-22和PBS作用于Ha Ca T细胞24小时后,提取细胞总蛋白,用Western blot法检测IL-22对细胞MAPK-ERK1/2和JAK/STAT这两条信号通路的影响;应用相关信号通路阻断剂PD98059和AG490与IL-22作用于Ha Ca T细胞后分别提取干预后细胞总蛋白及总m RNA,用Western blot法、ELISA法和实时荧光定量逆转录聚合酶链反应(RT-PCR)法检测其对Ha Ca T细胞分泌HB-EGF的影响。应用SPSS 17.0统计软件进行统计学处理,P0.05认为有显著性差异。2、应用咪喹莫特诱导小鼠背部皮肤产生银屑病样皮损,然后皮损处皮下注射1μg IL-22,48h后处死小鼠,取下背部皮肤,HE染色观察小鼠皮肤的改变,免疫组化观察HB-EGF的表达。3、用不同浓度(12.5μg/L、25μg/L、50μg/L、100μg/L)IL-22和应用相关信号通路阻断剂PD98059和AG490与IL-22处理Ha Ca T细胞,对照组用等量PBS处理,应用免疫荧光法检测细胞内TIG3蛋白的表达位置,分别提取干预后的细胞总蛋白及总m RNA,用Western blot法、ELISA法和实时荧光定量RT-PCR法检测其对Ha Ca T细胞分泌TIG3的影响。应用SPSS 17.0统计软件包进行统计学处理,P0.05认为有显著性差异。结果:1、IL-22可诱导HaCaT细胞MAPK信号通路中P-ERK1/2蛋白的增加和JAK2/STAT3信号通路中P-JAK2和P-STAT3蛋白的升高。加入信号通路特异性抑制剂PD98059及AG490后HB-EGF蛋白和m RNA的表达较IL-22干预组明显降低。2、咪喹莫特持续作用于Bal B/c小鼠背部,可以成功诱导小鼠银屑病样皮损,IL-22皮下注射可引起正常Bal B/c小鼠背部及银屑病小鼠皮肤表皮中HB-EGF表达的升高。3、IL-22可引起Ha Ca T细胞中TIG3蛋白水平的降低,其作用的强度与浓度有关:不同浓度IL-22处理组的细胞分泌TIG3的量均较对照组降低,且随着IL-22浓度的升高,其对TIG3表达的抑制作用逐渐增强。应用信号通路特异性抑制剂PD98059及AG490后IL-22对TIG3抑制作用较IL-22组减少。差异有统计学意义。结论:IL-22可以引起Ha Ca T细胞中MAPK-ERK1/2和JAK2/STAT3这两条信号通路激活。IL-22诱导Ha Ca T细胞产生HB-EGF蛋白的作用机制可能与MAPK-ERK1/2和JAK2/STAT3这两条信号通路有关。IL-22可引起Bal B/c小鼠背部正常皮肤及银屑病样皮损表皮中HB-EGF表达的升高。IL-22可以抑制Ha Ca T细胞中TIG3表达,抑制作用的强度与IL-22浓度有关,作用机制可能与MAPK-ERK1/2和JAK2/STAT3信号通路有关。
[Abstract]:Psoriasis is a common chronic recurrence of erythema and scaly disease. This disease can be divided into 4 types: common type, arthropathy, erythroderma type and pustular type. The pathogenesis is chronic and recurrent. The pathogenesis of this disease is not completely clear. At present, the pathogenesis of psoriasis is mainly attributed to auxiliary Th cells and related cell causes. An inflammatory response.Th22 cell targeting keratinocyte as a target is a newly discovered CD4+ auxiliary T cell subgroup, highly expressed in the peripheral blood of psoriasis, and its main cytokine is IL-22. through which the related biological functions are realized. In the peripheral blood and skin lesions of psoriasis patients, the IL-22 level and the severity of psoriasis are obvious. Our previous study shows that heparin binding to epidermal growth factor like growth factor (heparin-binding epidermal-growth-factor-like growth factor, HB-EGF) plays an important role in the abnormal proliferation and prognosis of epidermal KC in psoriasis. Previous studies in this study show that IL-22 can induce Ha Ca T cell expression in vitro. EGF, but the mechanism of action is not clear, the mitogen activated protein kinase (MAPK-ERK1/2) pathway and the transcription activating factor pathway (JAK/STAT) are the two signaling pathways that play an indispensable role in the pathogenesis of psoriasis. The gene 3 (Tazarotene-induced gene 3, TIG3) is a tumor suppressor,.TIG3, which regulates the keratinocyte The proliferation and differentiation of psoriasis and the decrease of expression in psoriasis. In summary, we speculate that IL-22 promoting KC secretion may be related to the two signaling pathways of MAPK-ERK1/2 and JAK/STAT, and that there is a certain relationship between TIG3 expression and.IL-22 by promoting HB-EGF expression and inhibiting the TIG3 expression to participate in the occurrence and development of silver chip disease. This experiment studies IL-22 promoting HaCaT thin. The mechanism of cell secretion of HB-EGF and its effect on the secretion of TIG3 by HaCaT cells. Objective: To study the mechanism of the action of interleukin 22 (Interleukin-22, IL-22) on the expression of HB-EGF in keratinocytes and the mechanism of its effect on the expression of TIG3. Methods: 1, IL-22 and 24 hours are used at different concentrations (12.5 mu g/L, 25 u g/L, 50 mu g/L, 100 mu g/L). After that, the total protein of cell was extracted and the effects of IL-22 on the two signal pathways of MAPK-ERK1/2 and JAK/STAT were detected by Western blot, and the total protein and total m were extracted after the action of PD98059 and AG490 and IL-22 on Ha Ca T cells. The effect of transcriptional polymerase chain reaction (RT-PCR) on the secretion of HB-EGF in Ha Ca T cells was detected. SPSS 17 statistical software was used for statistical processing. P0.05 found a significant difference in.2. Imiquimod induced psoriasis like skin lesions in the skin of mice on the back, and then subcutaneous injection of 1 UU IL-22,48h to kill the mice and take the lower back. Skin, HE staining was used to observe the changes in the skin of mice. The expression of HB-EGF was observed by immunohistochemical staining,.3, with the different concentrations (12.5 g/L, 25 g/L, 50, g/L, 100 g/L) IL-22 and the application related signaling pathway blockers, PD98059 and AG490 and IL-22. Location, the total protein and total m RNA were extracted, and the effect of Western blot, ELISA and real-time fluorescent quantitative RT-PCR on the secretion of TIG3 was detected by Ha Ca T cell. The SPSS 17 statistical package was used for statistical processing. The increase of K1/2 protein and the increase of P-JAK2 and P-STAT3 protein in the JAK2/STAT3 signaling pathway. The expression of HB-EGF protein and m RNA after adding signal pathway specific inhibitor PD98059 and AG4 90 is significantly lower than that in IL-22 intervention group. Imiquimod continues to act on the back of Bal mice, and can induce psoriasis like skin lesions in mice. The expression of HB-EGF in the skin epidermis of normal Bal B/c mice and psoriasis mice increased by.3. IL-22 could cause the decrease of TIG3 protein level in Ha Ca T cells. The intensity and concentration of its action were related to the concentration of TIG3 in the cells of different concentrations of IL-22 treatment group than that of the control group. The inhibitory effect of expression was gradually enhanced. The inhibitory effect of PD98059 and IL-22 on TIG3 decreased compared with the IL-22 group. The difference was statistically significant. Conclusion: IL-22 can cause MAPK-ERK1/2 and JAK2/STAT3 in Ha Ca T cells to induce two signaling pathways to induce the effect of.IL-22 cells. The mechanism may be related to the two signaling pathways of MAPK-ERK1/2 and JAK2/STAT3 that.IL-22 can cause the HB-EGF expression in the normal skin of Bal B/c mice and the HB-EGF expression in the epidermis of psoriasis like skin lesions. The.IL-22 can inhibit TIG3 expression in Ha Ca T cells. The inhibitory effect is related to the IL-22 concentration. The mechanism of action may be associated with the letter and the letter The number path is related.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R758.63

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