本文選題：細(xì)菌超抗原　切入點(diǎn)：SEB　出處：《第三軍醫(yī)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:炎癥性皮膚病是皮膚科最常見的一類疾病,包括特應(yīng)性皮炎、接觸性皮炎、濕疹、銀屑病等諸多皮膚疾病。針對(duì)這一類疾病的臨床治療,外用糖皮質(zhì)激素以其良好的抗炎、抗過敏、免疫抑制等作用,長久以來都是臨床醫(yī)師的主要選擇之一。但是,外用糖皮質(zhì)激素治療炎癥性皮膚病在取得一定療效的同時(shí),隨著用藥時(shí)間的延長,其療效會(huì)出現(xiàn)逐漸減弱的現(xiàn)象,此類現(xiàn)象被稱為外用糖皮質(zhì)激素抵抗(Topical glucocorticoid resistance)。炎癥性皮膚病的發(fā)病過程中,由于皮膚屏障功能的受損,細(xì)菌定植會(huì)增加,研究證實(shí)金黃色葡萄球菌所分泌的腸毒素B(Staphylococcal enterotoxin B,SEB)可能參與了外用糖皮質(zhì)激素抵抗的過程。探討SEB在外用糖皮質(zhì)激素抵抗發(fā)生中的作用及相關(guān)機(jī)制,對(duì)于炎癥性皮膚病外用糖皮質(zhì)激素制劑治療的規(guī)范、合理應(yīng)用,避免糖皮質(zhì)激素濫用現(xiàn)象,提高炎癥性皮膚病治療效果均有著重要的意義。研究目的:探討細(xì)菌超抗原SEB對(duì)角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體的影響及其相關(guān)機(jī)制。研究方法:選取人永生化角質(zhì)形成細(xì)胞(Ha Ca T細(xì)胞)作為細(xì)胞模型。以不同濃度的細(xì)菌超抗原SEB處理Ha Ca T細(xì)胞24 h后,采用RT-PCR方法檢測Ha Ca T細(xì)胞糖皮質(zhì)激素受體(Glucocorticoid receptor,GR)m RNA的表達(dá),western blot檢測Ha Ca T細(xì)胞GR的蛋白表達(dá)情況。以地塞米松作用Ha Ca T細(xì)胞不同時(shí)間點(diǎn),再以SEB預(yù)孵育Ha Ca T細(xì)胞后加入地塞米松作用細(xì)胞,免疫熒光法在激光共聚焦顯微鏡下觀察Ha Ca T細(xì)胞內(nèi)糖皮質(zhì)激素受體GRα的分布情況。之后,以SEB作用Ha Ca T細(xì)胞,western blot檢測細(xì)胞內(nèi)MAPK信號(hào)通路活性,再加入通路抑制劑觀察SEB作用Ha Ca T細(xì)胞后對(duì)GR蛋白表達(dá)的影響及GRα的細(xì)胞內(nèi)分布情況。研究結(jié)果:細(xì)菌超抗原SEB對(duì)角質(zhì)形成細(xì)胞糖皮質(zhì)激素受體m RNA及蛋白表達(dá)的影響:(1)細(xì)菌超抗原SEB作用Ha Ca T細(xì)胞后,與對(duì)照組相比,其糖皮質(zhì)激素受體GRα的m RNA表達(dá)無統(tǒng)計(jì)學(xué)差異(p0.05);(2)細(xì)菌超抗原SEB作用Ha Ca T細(xì)胞后,與對(duì)照組相比,其糖皮質(zhì)激素受體GRβ的m RNA表達(dá)升高,且在SEB濃度為100 ng/ml時(shí)作用最強(qiáng);(3)細(xì)菌超抗原SEB作用Ha Ca T細(xì)胞后,與對(duì)照組相比,其糖皮質(zhì)激素受體GRα的蛋白表達(dá)無統(tǒng)計(jì)學(xué)差異(p0.05);(4)細(xì)菌超抗原SEB作用Ha Ca T細(xì)胞后,與對(duì)照組相比,其糖皮質(zhì)激素受體GRβ的蛋白表達(dá)升高,且在SEB濃度為100 ng/ml時(shí)作用最強(qiáng)。2.細(xì)菌超抗原SEB對(duì)Ha Ca T細(xì)胞糖皮質(zhì)激素受體GRα核轉(zhuǎn)位的影響:(1)10-6mol/L地塞米松作用Ha Ca T細(xì)胞8 h后,能夠誘導(dǎo)GRα從胞質(zhì)向胞核內(nèi)轉(zhuǎn)位,并且,該效應(yīng)在地塞米松作用Ha Ca T細(xì)胞24 h后仍然存在;(2)先以100 ng/ml的SEB預(yù)孵育Ha Ca T細(xì)胞1 h,再以10-6 mol/L地塞米松作用Ha Ca T細(xì)胞8 h后,SEB組與空白對(duì)照組相比,兩組細(xì)胞內(nèi)GRα主要分布在細(xì)胞胞質(zhì)中;與空白對(duì)照組相比,地塞米松組Ha Ca T細(xì)胞內(nèi)GRα的分布出現(xiàn)向胞核內(nèi)轉(zhuǎn)位的現(xiàn)象,地塞米松+SEB組細(xì)胞中GRα的分布仍主要局限于胞質(zhì)中,向胞核內(nèi)轉(zhuǎn)位的效應(yīng)顯著減低。細(xì)菌超抗原SEB對(duì)角質(zhì)形成細(xì)胞內(nèi)MAPK信號(hào)通路的作用:(1)細(xì)菌超抗原SEB作用Ha Ca T細(xì)胞后,細(xì)胞內(nèi)p38信號(hào)通路未見激活;(2)細(xì)菌超抗原SEB作用Ha Ca T細(xì)胞后,細(xì)胞內(nèi)JNK信號(hào)通路未見激活;(3)細(xì)菌超抗原SEB作用Ha Ca T細(xì)胞后,可激活細(xì)胞內(nèi)ERK1/2信號(hào)通路;(4)加入ERK1/2抑制劑U0126后,以SEB作用Ha Ca T細(xì)胞,與對(duì)照組相比,糖皮質(zhì)激素受體GRβ的蛋白表達(dá)無統(tǒng)計(jì)學(xué)差異(p0.05);(5)先以100 ng/ml的SEB+ERK抑制劑U0126預(yù)孵育Ha Ca T細(xì)胞1 h,再以10-6mol/L地塞米松作用Ha Ca T細(xì)胞8 h后,與空白對(duì)照組相比,地塞米松組、地塞米松+SEB+U0126組Ha Ca T細(xì)胞內(nèi)GRα的分布均出現(xiàn)向胞核內(nèi)轉(zhuǎn)位的現(xiàn)象。結(jié)論:1.細(xì)菌超抗原SEB作用對(duì)Ha Ca T細(xì)胞糖激素受體GRα的m RNA和蛋白表達(dá)水平無顯著影響,但是能上調(diào)GRβ的m RNA和蛋白表達(dá),并且在SEB濃度為100 ng/ml時(shí)作用最強(qiáng);2.地塞米松可以誘導(dǎo)Ha Ca T細(xì)胞GRα從胞質(zhì)向胞核內(nèi)轉(zhuǎn)位,而SEB可以抑制地塞米松誘導(dǎo)的Ha Ca T細(xì)胞GRα由胞質(zhì)向胞核轉(zhuǎn)位的效應(yīng);3.細(xì)菌超抗原SEB可以激活細(xì)胞內(nèi)ERK1/2信號(hào)通路,抑制該通路活性后,SEB對(duì)Ha Ca T細(xì)胞GRβ蛋白表達(dá)的上調(diào)作用受到抑制,同時(shí),SEB對(duì)地塞米松誘導(dǎo)的Ha Ca T細(xì)胞GRα由胞質(zhì)向胞核轉(zhuǎn)位的抑制作用顯著減弱。
[Abstract]:Background: inflammatory skin disease is one of the most common diseases in Department of Dermatology, including atopic dermatitis, contact dermatitis, eczema, psoriasis and other skin diseases. The clinical treatment for this disease, topical corticosteroids with good anti-inflammatory, anti allergy, immune suppression and so on, for a long time is one of the main choice in clinic. However, topical corticosteroids in the treatment of inflammatory skin diseases have certain curative effect at the same time, along with the prolongation of time, the effect will gradually weaken the phenomenon, this phenomenon is called topical glucocorticoid resistance (Topical glucocorticoid resistance). The pathogenesis of inflammatory skin disease, due to impaired skin barrier function, bacterial colonization will increase, studies have confirmed that secreted by Staphylococcus aureus enterotoxin B (Staphylococcal enterotoxin B, SEB) may be involved in the use of sugar The process of glucocorticoid resistance. To investigate SEB in the genesis of the effect and mechanism of topical glucocorticoid resistance, for the treatment of inflammatory skin disease with topical corticosteroid preparation standard, reasonable application, avoid the abuse of glucocorticoid, improve the therapeutic effect of inflammatory skin disease has important significance. Objective: To investigate the effect bacterial superantigen SEB on keratinocytes of glucocorticoid receptor and its mechanism. Methods: selected human immortalized keratinocyte (Ha Ca T cells) as a cell model with different concentrations of bacterial superantigen SEB Ha Ca T 24 h Ha Ca cells, T cells were detected by glucocorticoid receptor RT-PCR (Glucocorticoid receptor GR) expression of M RNA, Western blot Ha Ca T expression detection cell GR protein. In different time point Ha Ca dexamethasone T cells, followed by SEB pre Ha Ca after T cells were incubated with dexamethasone in cells, immunofluorescence method was used to observe the distribution of Ha Ca in T cells GR of glucocorticoid receptor alpha under confocal microscopy. Then, the effect of SEB Ha Ca T MAPK cells, Western signaling pathway activity detection of blot cells, the distribution of effects on expression of GR protein join SEB Ha Ca pathway inhibitors were observed after T cells and GR cells. The alpha results: bacterial superantigen SEB cells influence the formation of sugar expression of glucocorticoid receptor m and RNA protein in keratinocytes: (1) bacterial superantigen SEB Ha Ca T cells, compared with control group, m RNA the glucocorticoid receptor alpha GR expression had no statistical difference (P0.05); (2) bacterial superantigen SEB Ha Ca T cells, compared with the control group, the m RNA GR of glucocorticoid receptor beta expression increased, and the concentration of SEB was 100 ng/ ml had the strongest effect; (3) 緇嗚弻瓚呮姉鍘烻EB浣滅敤Ha Ca T緇嗚優(yōu)鍚，

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