本文選題：變應性接觸性皮炎 + SEB�。� 參考：《第三軍醫(yī)大學》2016年碩士論文

【摘要】：研究背景和目的外用糖皮質(zhì)激素(glucocorticoids,GC)作為多種皮膚病的治療手段已有較長歷史。然而,在臨床工作中常發(fā)現(xiàn)許多炎癥性皮膚病患者長期外用GC后出現(xiàn)GC療效下降,這種現(xiàn)象被稱為GC的“快速減敏(tachyphylaxis)”或“快速耐受(acute tolerance)”。研究發(fā)生激素抵抗的機制對于改善外用GC療效至關重要。長期外用GC的皮膚病如銀屑病、特應性皮炎通常伴隨金黃色葡萄球菌定植,金黃色葡萄球菌能分泌或者代謝具有免疫原性的外毒素——細菌超抗原,如金黃色葡萄球菌腸毒素B(staphylococcal enterotoxin B,SEB)。SEB不需要抗原呈遞細胞加工,即能被T細胞識別,激活大量T細胞(大約占總T細胞的20%),導致TNF-α、TNF-β、IL-2和INF-γ等炎癥因子大量釋放,從而加重各種組織的炎性和過敏反應。研究已經(jīng)發(fā)現(xiàn)SEB能誘導系統(tǒng)激素抵抗。我們前期實驗結(jié)果顯示SEB可能上調(diào)皮炎局部糖皮質(zhì)激素受體β(glucocorticoid receptorβ,GRβ)表達介導激素抵抗,但SEB是否影響角質(zhì)形成細胞GRα核轉(zhuǎn)移尚不清楚,本研究將建立小鼠變應性接觸性皮炎(allergic contact dermatitis,ACD)模型,觀察SEB是否抑制角質(zhì)形成細胞GRα核轉(zhuǎn)移,探討FKBP51和FKBP52表達是否影響SEB對GRα核轉(zhuǎn)移的作用,以及他克莫司是否能拮抗SEB對外用GC的抵抗作用,旨在提出SEB導致外用激素抵抗的新機制和新認識,為今后找到可能的治療靶點提供了新的實驗依據(jù)。方法使用1%DNCB建立小鼠變應性接觸性皮炎(allergic contact dermatitis,ACD)模型,采用HE染色檢測小鼠背部皮膚炎性細胞數(shù)變化;激光共聚焦定位GRα在小鼠角質(zhì)形成細胞中的分布;Western blot檢測GRα在小鼠角質(zhì)形成細胞胞漿和胞核的表達;ELISA檢測各組IL-2、4、13和TNF-a炎癥因子的表達;Real-Time PCR和Western blot分別檢測FK506結(jié)合蛋白51(FK506-binding protein 51,FKBP51)和FKBP52 m RNA和蛋白的表達。實驗分2部分進行探討:1)細菌超抗原SEB抑制小鼠角質(zhì)形成細胞糖皮質(zhì)激素受體核轉(zhuǎn)移作用及機制研究;2)他克莫司、FKBP51和FKBP52調(diào)控SEB小鼠角質(zhì)形成細胞糖皮質(zhì)受體核轉(zhuǎn)移障礙的作用及機制。結(jié)果1.與皮炎組相比,SEB處理皮炎組真皮內(nèi)炎性細胞顯著增加。與地塞米松處理組相比,SEB可減少地塞米松抗炎作用,SEB的抑制作用可被他克莫司拮抗;2.與皮炎組相比,地塞米松處理皮炎組GRα核/漿陽性比值顯著增高。與地塞米松處理皮炎組相比,SEB可顯著降低SEB+地塞米松處理組GRα核/漿陽性比值,但他克莫司可拮抗SEB,提高GRα核/漿陽性比值。3.與皮炎組相比,地塞米松可顯著增加胞漿GRα蛋白表達。與地塞米松處理組相比,SEB可顯著增加胞漿GRα滯留量,減少GRα入核量。SEB對GRα核轉(zhuǎn)移抑制可被他克莫司部分拮抗。4.與皮炎組相比,地塞米松處理皮炎組IL-2、4、13和TNF-a炎癥因子顯著減少。與地塞米松處理組相比,SEB可顯著拮抗地塞米松對炎性因子的抑制作用,但他克莫司可拮抗SEB這一作用;5.與正常對照組相比,地塞米松處理組角質(zhì)形成細胞FKBP51明顯增高,而FKBP52無影響,提示地塞米松可能通過提高FKBP51表達負調(diào)控自身的抗炎作用。SEB處理后也顯著增加FKBP51表達,而對FKBP52無影響,提示SEB導致的激素抵抗作用可能是通過上調(diào)FKBP51表達水平來實現(xiàn)的。而他克莫司處理后,可顯著降低SEB和地塞米松對FKBP51的上調(diào)作用,提示他克莫司抗炎作用可能部分是通過減少FKBP51表達實現(xiàn)的。全文結(jié)論1.SEB能減弱地塞米松對小鼠變應性接觸性皮炎的抗炎作用;2.SEB誘導的激素抵抗與其阻礙角質(zhì)形成細胞GRα核轉(zhuǎn)移有關;3.SEB誘導GRα核轉(zhuǎn)移障礙與其增加角質(zhì)形成細胞FKBP51表達有關;4.他克莫司能恢復SEB減弱的激素療效,機制與其下調(diào)SEB誘導的FKBP51表達和逆轉(zhuǎn)SEB介導的GRα核轉(zhuǎn)移障礙有關。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R751

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