细菌超抗原SEB抑制小鼠角质形成细胞糖皮质激素受体核转移作用及机制研究

发布时间：2018-07-06 19:01

本文选题：变应性接触性皮炎 + SEB　；参考：《第三军医大学》2016年硕士论文

【摘要】：研究背景和目的外用糖皮质激素(glucocorticoids,GC)作为多种皮肤病的治疗手段已有较长历史。然而,在临床工作中常发现许多炎症性皮肤病患者长期外用GC后出现GC疗效下降,这种现象被称为GC的“快速减敏(tachyphylaxis)”或“快速耐受(acute tolerance)”。研究发生激素抵抗的机制对于改善外用GC疗效至关重要。长期外用GC的皮肤病如银屑病、特应性皮炎通常伴随金黄色葡萄球菌定植,金黄色葡萄球菌能分泌或者代谢具有免疫原性的外毒素——细菌超抗原,如金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)。SEB不需要抗原呈递细胞加工,即能被T细胞识别,激活大量T细胞(大约占总T细胞的20%),导致TNF-α、TNF-β、IL-2和INF-γ等炎症因子大量释放,从而加重各种组织的炎性和过敏反应。研究已经发现SEB能诱导系统激素抵抗。我们前期实验结果显示SEB可能上调皮炎局部糖皮质激素受体β(glucocorticoid receptorβ,GRβ)表达介导激素抵抗,但SEB是否影响角质形成细胞GRα核转移尚不清楚,本研究将建立小鼠变应性接触性皮炎(allergic contact dermatitis,ACD)模型,观察SEB是否抑制角质形成细胞GRα核转移,探讨FKBP51和FKBP52表达是否影响SEB对GRα核转移的作用,以及他克莫司是否能拮抗SEB对外用GC的抵抗作用,旨在提出SEB导致外用激素抵抗的新机制和新认识,为今后找到可能的治疗靶点提供了新的实验依据。方法使用1%DNCB建立小鼠变应性接触性皮炎(allergic contact dermatitis,ACD)模型,采用HE染色检测小鼠背部皮肤炎性细胞数变化;激光共聚焦定位GRα在小鼠角质形成细胞中的分布;Western blot检测GRα在小鼠角质形成细胞胞浆和胞核的表达;ELISA检测各组IL-2、4、13和TNF-a炎症因子的表达;Real-Time PCR和Western blot分别检测FK506结合蛋白51(FK506-binding protein 51,FKBP51)和FKBP52 m RNA和蛋白的表达。实验分2部分进行探讨:1)细菌超抗原SEB抑制小鼠角质形成细胞糖皮质激素受体核转移作用及机制研究;2)他克莫司、FKBP51和FKBP52调控SEB小鼠角质形成细胞糖皮质受体核转移障碍的作用及机制。结果1.与皮炎组相比,SEB处理皮炎组真皮内炎性细胞显著增加。与地塞米松处理组相比,SEB可减少地塞米松抗炎作用,SEB的抑制作用可被他克莫司拮抗;2.与皮炎组相比,地塞米松处理皮炎组GRα核/浆阳性比值显著增高。与地塞米松处理皮炎组相比,SEB可显著降低SEB+地塞米松处理组GRα核/浆阳性比值,但他克莫司可拮抗SEB,提高GRα核/浆阳性比值。3.与皮炎组相比,地塞米松可显著增加胞浆GRα蛋白表达。与地塞米松处理组相比,SEB可显著增加胞浆GRα滞留量,减少GRα入核量。SEB对GRα核转移抑制可被他克莫司部分拮抗。4.与皮炎组相比,地塞米松处理皮炎组IL-2、4、13和TNF-a炎症因子显著减少。与地塞米松处理组相比,SEB可显著拮抗地塞米松对炎性因子的抑制作用,但他克莫司可拮抗SEB这一作用;5.与正常对照组相比,地塞米松处理组角质形成细胞FKBP51明显增高,而FKBP52无影响,提示地塞米松可能通过提高FKBP51表达负调控自身的抗炎作用。SEB处理后也显著增加FKBP51表达,而对FKBP52无影响,提示SEB导致的激素抵抗作用可能是通过上调FKBP51表达水平来实现的。而他克莫司处理后,可显著降低SEB和地塞米松对FKBP51的上调作用,提示他克莫司抗炎作用可能部分是通过减少FKBP51表达实现的。全文结论1.SEB能减弱地塞米松对小鼠变应性接触性皮炎的抗炎作用;2.SEB诱导的激素抵抗与其阻碍角质形成细胞GRα核转移有关;3.SEB诱导GRα核转移障碍与其增加角质形成细胞FKBP51表达有关;4.他克莫司能恢复SEB减弱的激素疗效,机制与其下调SEB诱导的FKBP51表达和逆转SEB介导的GRα核转移障碍有关。
[Abstract]:Background and objective glucocorticoids (GC) has a long history as a treatment for multiple dermatosis. However, in clinical work, many patients with inflammatory dermatoses are often found to have a decrease in GC after long-term external use of GC. This phenomenon is called "rapid desensitization (tachyphylaxis)" or "fast tolerance (AC)" (AC). Ute tolerance) ". The study of the mechanism of hormone resistance is essential to improve the efficacy of external GC. Long-term external use of GC skin diseases such as psoriasis, atopic dermatitis usually accompanied by Staphylococcus aureus colonization, Staphylococcus aureus can secrete or metabolize an immunogenic exotoxin - bacterial superantigen, such as golden yellow globules The bacterial enterotoxin B (staphylococcal enterotoxin B, SEB).SEB does not need antigen presenting cell processing, that is, it can be identified by T cells and activates a large number of T cells (about 20% of the total T cells), leading to a large release of inflammatory factors such as TNF- a, TNF- beta, IL-2 and gamma, which aggravates the inflammatory and allergic reactions of various tissues. Our preliminary results show that SEB may mediate hormone resistance in local glucocorticoid receptor beta (glucocorticoid receptor beta, GR beta), but whether SEB affects GR alpha nuclear transfer in keratinocytes is not clear. This study will establish a mouse allergic contact dermatitis (allergic contact dermatitis, ACD). To observe whether SEB inhibits keratinocyte GR alpha nuclear transfer, and explores whether the expression of FKBP51 and FKBP52 affects the effect of SEB on GR alpha nuclear transfer, and whether tacrolimus can antagonize the resistance of SEB to external GC, and aims to propose a new mechanism and new understanding of SEB leading to the resistance of external use hormone, and to provide a potential therapeutic target for the future. New experimental basis. Methods the model of mouse allergic contact dermatitis (allergic contact dermatitis, ACD) was established by 1%DNCB. The number of inflammatory cells in the back skin of mice was detected by HE staining, and the distribution of GR alpha in the keratinocytes of mice was localized by laser confocal microscopy; Western blot detected GR a in the cytoplasm and cell of mouse keratinocytes. The expression of IL-2,4,13 and TNF-a inflammatory factors were detected by ELISA; Real-Time PCR and Western blot were used to detect the expression of FK506 binding protein 51 (FK506-binding protein 51, FKBP51) and FKBP52 proteins and proteins. The experiment was divided into 2 parts: 1) bacterial superantigen was used to inhibit the corticosteroid receptor nucleus of mouse keratinocytes Transfer and mechanism study; 2) tacrolimus, FKBP51 and FKBP52 regulated the effect and mechanism of the nuclear transfer disorder in the keratinocytes of SEB mice. Results compared with the dermatitis group 1., the dermatitis intradermal inflammatory cells in the dermatitis group were significantly increased. Compared with the dexamethasone treatment group, SEB could reduce the anti-inflammatory effect of dexamethasone and the inhibition of SEB. The effect can be antagonized by tacrolimus; 2. compared with dermatitis, the positive ratio of GR alpha nucleus / pulp in dexamethasone treated dermatitis group was significantly higher. Compared with dexamethasone treated dermatitis group, SEB could significantly reduce the GR alpha core / pulp positive ratio of SEB+ dexamethasone treatment group, but tacrolimus can antagonize SEB and improve the GR alpha core / pulp positive ratio.3. compared with dermatitis group. Dexamethasone significantly increased the expression of cytoplasmic GR alpha protein. Compared with the dexamethasone treatment group, SEB significantly increased the retention of cytoplasmic GR a, and reduced the GR alpha nucleation.SEB on GR alpha nuclear transfer inhibition could be partially antagonized by tacrolimus in.4. with the dermatitis group, and dexamethasone treated Pi Yanzu IL-2,4,13 and TNF-a inflammatory factors significantly. Compared with the pine treatment group, SEB could significantly antagonize the inhibition of dexamethasone on inflammatory factors, but tacrolimus could antagonize the effect of SEB. 5. compared with the normal control group, the keratinocyte FKBP51 in the dexamethasone treatment group was significantly higher, while FKBP52 had no effect, suggesting that dexamethasone might improve the anti inflammatory activity of the FKBP51 expression by increasing the negative regulation of the FKBP51 expression. .SEB treatment also significantly increased the expression of FKBP51, but had no effect on FKBP52, suggesting that SEB induced hormone resistance may be achieved by up regulation of FKBP51 expression. And tacrolimus can significantly reduce the effect of SEB and dexamethasone on FKBP51, suggesting that the anti inflammatory effect of the camosi may be partly by reducing FKBP5. 1 expression is realized. Conclusion 1.SEB can weaken the anti-inflammatory effect of dexamethasone on allergic contact dermatitis in mice; 2.SEB induced hormone resistance is related to its obstruction of keratinocyte GR alpha nuclear transfer; 3.SEB induced GR alpha metastasis is related to the increase of FKBP51 surface of keratinocytes; and tacrolimus can restore the excitation of SEB weakening. The mechanism of its effect is related to its down-regulation of SEB induced FKBP51 expression and the reversal of SEB mediated GR alpha nuclear transfer dysfunction.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R751

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