细菌超抗原SEB对角质形成细胞糖皮质激素受体的影响及机制研究

发布时间：2018-03-07 10:13

  本文选题：细菌超抗原　切入点：SEB　出处：《第三军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景:炎症性皮肤病是皮肤科最常见的一类疾病,包括特应性皮炎、接触性皮炎、湿疹、银屑病等诸多皮肤疾病。针对这一类疾病的临床治疗,外用糖皮质激素以其良好的抗炎、抗过敏、免疫抑制等作用,长久以来都是临床医师的主要选择之一。但是,外用糖皮质激素治疗炎症性皮肤病在取得一定疗效的同时,随着用药时间的延长,其疗效会出现逐渐减弱的现象,此类现象被称为外用糖皮质激素抵抗(Topical glucocorticoid resistance)。炎症性皮肤病的发病过程中,由于皮肤屏障功能的受损,细菌定植会增加,研究证实金黄色葡萄球菌所分泌的肠毒素B(Staphylococcal enterotoxin B,SEB)可能参与了外用糖皮质激素抵抗的过程。探讨SEB在外用糖皮质激素抵抗发生中的作用及相关机制,对于炎症性皮肤病外用糖皮质激素制剂治疗的规范、合理应用,避免糖皮质激素滥用现象,提高炎症性皮肤病治疗效果均有着重要的意义。研究目的:探讨细菌超抗原SEB对角质形成细胞糖皮质激素受体的影响及其相关机制。研究方法:选取人永生化角质形成细胞(Ha Ca T细胞)作为细胞模型。以不同浓度的细菌超抗原SEB处理Ha Ca T细胞24 h后,采用RT-PCR方法检测Ha Ca T细胞糖皮质激素受体(Glucocorticoid receptor,GR)m RNA的表达,western blot检测Ha Ca T细胞GR的蛋白表达情况。以地塞米松作用Ha Ca T细胞不同时间点,再以SEB预孵育Ha Ca T细胞后加入地塞米松作用细胞,免疫荧光法在激光共聚焦显微镜下观察Ha Ca T细胞内糖皮质激素受体GRα的分布情况。之后,以SEB作用Ha Ca T细胞,western blot检测细胞内MAPK信号通路活性,再加入通路抑制剂观察SEB作用Ha Ca T细胞后对GR蛋白表达的影响及GRα的细胞内分布情况。研究结果:细菌超抗原SEB对角质形成细胞糖皮质激素受体m RNA及蛋白表达的影响:(1)细菌超抗原SEB作用Ha Ca T细胞后,与对照组相比,其糖皮质激素受体GRα的m RNA表达无统计学差异(p0.05);(2)细菌超抗原SEB作用Ha Ca T细胞后,与对照组相比,其糖皮质激素受体GRβ的m RNA表达升高,且在SEB浓度为100 ng/ml时作用最强;(3)细菌超抗原SEB作用Ha Ca T细胞后,与对照组相比,其糖皮质激素受体GRα的蛋白表达无统计学差异(p0.05);(4)细菌超抗原SEB作用Ha Ca T细胞后,与对照组相比,其糖皮质激素受体GRβ的蛋白表达升高,且在SEB浓度为100 ng/ml时作用最强。2.细菌超抗原SEB对Ha Ca T细胞糖皮质激素受体GRα核转位的影响:(1)10-6mol/L地塞米松作用Ha Ca T细胞8 h后,能够诱导GRα从胞质向胞核内转位,并且,该效应在地塞米松作用Ha Ca T细胞24 h后仍然存在;(2)先以100 ng/ml的SEB预孵育Ha Ca T细胞1 h,再以10-6 mol/L地塞米松作用Ha Ca T细胞8 h后,SEB组与空白对照组相比,两组细胞内GRα主要分布在细胞胞质中;与空白对照组相比,地塞米松组Ha Ca T细胞内GRα的分布出现向胞核内转位的现象,地塞米松+SEB组细胞中GRα的分布仍主要局限于胞质中,向胞核内转位的效应显著减低。细菌超抗原SEB对角质形成细胞内MAPK信号通路的作用:(1)细菌超抗原SEB作用Ha Ca T细胞后,细胞内p38信号通路未见激活;(2)细菌超抗原SEB作用Ha Ca T细胞后,细胞内JNK信号通路未见激活;(3)细菌超抗原SEB作用Ha Ca T细胞后,可激活细胞内ERK1/2信号通路;(4)加入ERK1/2抑制剂U0126后,以SEB作用Ha Ca T细胞,与对照组相比,糖皮质激素受体GRβ的蛋白表达无统计学差异(p0.05);(5)先以100 ng/ml的SEB+ERK抑制剂U0126预孵育Ha Ca T细胞1 h,再以10-6mol/L地塞米松作用Ha Ca T细胞8 h后,与空白对照组相比,地塞米松组、地塞米松+SEB+U0126组Ha Ca T细胞内GRα的分布均出现向胞核内转位的现象。结论:1.细菌超抗原SEB作用对Ha Ca T细胞糖激素受体GRα的m RNA和蛋白表达水平无显著影响,但是能上调GRβ的m RNA和蛋白表达,并且在SEB浓度为100 ng/ml时作用最强;2.地塞米松可以诱导Ha Ca T细胞GRα从胞质向胞核内转位,而SEB可以抑制地塞米松诱导的Ha Ca T细胞GRα由胞质向胞核转位的效应;3.细菌超抗原SEB可以激活细胞内ERK1/2信号通路,抑制该通路活性后,SEB对Ha Ca T细胞GRβ蛋白表达的上调作用受到抑制,同时,SEB对地塞米松诱导的Ha Ca T细胞GRα由胞质向胞核转位的抑制作用显著减弱。
[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) 缁嗚弻瓒呮姉鍘烻EB浣滅敤Ha Ca T缁嗚優鍚，

本文编号：1579016