【作者】 郁博；

【导师】 何威；

【作者基本信息】 第三军医大学 ， 皮肤性病学， 2008， 博士

【摘要】 背景糖皮质激素外用制剂是治疗炎症性皮肤病的主要药物。炎症性皮肤病患者使用糖皮质激素外用制剂易出现快速减敏和治疗抵抗等现象。然而,这些现象的确切机理尚不清楚。糖皮质激素(GC)通过与细胞内的糖皮质激素受体(GR)结合而发挥作用。它们结合后形成二聚体结构并转位进入细胞核,进而发挥转录因子的作用。人类GR分为两种亚型:GRα和GRβ,它们起源于GR前转录物的选择性剪切。共转染检测表明GRβ通过涉及GRα/GRβ异二聚体形成的机制而具有GRα介导转录激活的显性负相抑制子的功能。这些异二聚体的形成可能解释细胞中高表达GRβ导致的激素作用效应降低的现象。GRβ对GRα活性抑制作用产生了这样一种假说:多种炎症性疾病中GRβ相对于GRα的过度表达可能扮演着细胞对激素敏感性调控的作用。GR和核因子-κB (NF-κB)是免疫及炎症反应中两个重要的调节因素,但它们却起着完全相反的调节作用。NF-κB可介导多种与免疫和炎症相关的细胞因子和介质的活化过程,而GR则可抑制NF-κB活化基因的表达。此外,肿瘤坏死因子-α(TNF-α)也是重要的炎性细胞因子,通过激活它们的细胞内信号通路,积极参与皮肤炎症的病理生理过程。我们的前期研究曾发现慢性皮炎表皮中GRα表达下调。然而,迄今为止,对于角质形成细胞而言,糖皮质激素对其GR和NF-κB/IκB的调节作用尚不明确。TNF-α在多种炎症性皮肤病皮损局部表达增高,在人角质形成细胞中,它对GR的表达和对NF-κB/IκB的调节作用也不清楚。皮损局部表皮GR和NF-κB/IκB的调节变化是否与外用糖皮质激素所出现的快速减敏和治疗抵抗现象有关也值得进一步探索。目的探讨糖皮质激素对角质形成细胞的糖皮质激素受体(GRα和GRβ)的表达、NF-κB活性及IκBα表达的调节作用;比较三种不同的糖皮质激素(氢化可的松、地塞米松、倍他米松)对角质形成细胞的糖皮质激素受体和NF-κB/IκBα调节效应的差异;探讨TNF-α对角质形成细胞的糖皮质激素受体(GRα和GRβ)表达、NF-κB活性及IκBα表达的调节作用。旨在深入理解糖皮质激素和TNF-α对角质形成细胞GRα/GRβ、NF-κB/IκBα的调节作用,为正确使用糖皮质激素外用制剂治疗炎症性皮肤病提供理论和实验依据,对阐明糖皮质激素外用制剂易出现快速减敏和治疗抵抗现象的内在机制具有积极意义。方法与结果1、应用SP法免疫组化技术检测慢性皮炎(慢性湿疹和慢性单纯性苔藓)与正常对照皮肤中GRα的的表达情况。结果表明,与正常对照皮肤相比,慢性皮炎中GRα的表达显著下调。2、采用反转录Real-Time PCR和Western blotting,观察经不同浓度及不同时间的地塞米松作用下,培养的人角质形成细胞HaCaT细胞的GRα、GRβ、IκB-αmRNA和蛋白质的表达;用Western blotting法检测NF-κB核蛋白的表达情况。结果显示,地塞米松对HaCaT细胞GRα与CRβ的表达有不同的调节作用。地塞米松可下调HaCaT细胞GRα的表达,呈作用时间依赖性效应。同时,地塞米松可上调HaCaT细胞GRβ的表达,呈作用浓度依赖性效应和时间依赖性效应。地塞米松可诱导HaCaT细胞IκBα的表达而抑制NF-κB核转位,此作用呈浓度依赖性效应,在36h作用最明显。3、将培养的人角质形成细胞HaCaT细胞分为空白对照组、氢化可的松作用组、地塞米松作用组、倍他米松作用组,采用反转录Real-Time PCR和Western blotting法检测各组HaCaT细胞的GRα、GRβ、IκBαmRNA和蛋白质表达,用Western blotting法检测NF-κB核蛋白的表达情况。结果显示,三种激素均具有下调HaCaT细胞GRα表达的作用,其中氢化可的松作用最弱,而地塞米松与倍他米松作用之间无明显差异;三种激素均具有上调HaCaT细胞GRβ表达的作用,但此作用之间无明显差异。三种激素均具有诱导HaCaT细胞IκBα的表达而抑制NF-κB核转位的作用,其中氢化可的松作用最弱,而地塞米松与倍他米松作用之间无明显差异。4、采用反转录Real-Time PCR和Western blotting,观察经不同浓度及不同时间的肿瘤坏死因子(TNF-α)作用下,培养的人角质形成细胞HaCaT细胞的GRα、GRβ、IκBαmRNA和蛋白质表达,用Western blotting法检测NF-κB核蛋白的表达情况。结果显示,TNF-α对HaCaT细胞GRα的表达无明显影响,但可上调HaCaT细胞GRβ的表达,并且呈剂量和时间依赖性的上调趋势。TNF-α可抑制HaCaT细胞IκBα的表达而诱导NF-κB的核转位,呈作用浓度依赖性效应,此作用在1h达到高峰,此后逐渐减弱。结论1、与正常对照皮肤相比,慢性皮炎中GRα表达的下调可能会干扰皮损局部的抗炎机制并限制外用激素的疗效。2、糖皮质激素对角质形成细胞GRα和GRβ表达的不同调节作用可能部分解释外用糖皮质激素制剂的皮肤耐受现象和对糖皮质激素的抵抗。糖皮质激素通过调控人角质形成细胞NF-κB/IκB信号通路,可抑制皮损局部的炎症反应。3、不同效力的糖皮质激素对角质形成细胞GRα和GRβ的调节效力不同,对角质形成细胞NF-κB/IκB的调节效力也不同。这提示了临床上正确选用不同效力糖皮质激素外用制剂的必要性。4、TNF-α不但可通过调控角质形成细胞NF-κB/IκB可参与皮肤的炎症反应,而且还可诱导角质形成细胞GRβ的过度表达。后者可能是炎性细胞因子参与诱发糖皮质激素皮肤耐受现象的内在机制之一。更多还原

【Abstract】 BackgroundTopical glucocorticoid therapy is mainly a routine therapeutic modality for the treatment of inflammatory skin deseases. Tachyphylaxis and resistance to topical glucocorticoid agents often occur during the treatment of inflammatory skin deseases. However, the mechanisms of tachyphylaxis and resistance to topical glucocorticoid therapy are unknown.Glucocorticoids work by binding to the cytosolic glucocorticoid receptor (GR), inducing the formation of a dimer that is translocated into the nucleus and acts as a transcription factor. Cloning of the human GR has identified two isoforms, termed GRαand GRβ, which originate from alternative splicing of the GR primary transcript. Co-transfection assays have shown GRβto function as a dominant-negative inhibitor of GRα–mediated transcriptional activation through a mechanism that involves the formation of GRα/GRβheterodimers. Such heterodimer formation may account for the reduced effectiveness of glucocorticoid action in cells overexpressing GRβ. This inhibitory effect of GRβon GRαactivity led to the hypothesis that excessive expression of the GRβisoform with respect to GRαmight play a role in the regulation of a cell’s sensitivity to glucocorticoids in various inflammatory diseases.GR and nuclear factorκB (NF-κB) are both important regulatory factors, with entirely opposite effects, involved in immune response and inflammation. NF-κB plays a role in activating transcription of many cytokines and mediators associated with immune response or inflammation while GR may inhibit gene expression of many cytokines and mediators activated by NF-κB. Moreover, TNF-αis important inflammatory cytokines actively participating in pathophysiological process of skin inflammation via activating their intracellular signaling pathway.Our previous studies has shown the downregulated expression of GRαin chronic dermatitis. However, effects of glucocorticoid on expression of glucocorticoid receptor and NF-κB/IκB in human keratinocytes remain unknown up till now. Effects of overexpressing TNF-αin local skin of inflammatory skin deseases on expression of glucocorticoid receptor and NF-κB/IκB in human keratinocytes are unclear also. Whether do they associated with phenomenon of tachyphylaxis and resistance to topical glucocorticoid therapy in inflammatory skin deseases?ObjectiveTo reconfirm expression of GRαin chronic dermatitides.To investigate Effects of dexamethasone on expression of glucocorticoid receptor and regulation of NF-κB/IκB in human keratinocyte HaCaT cell line, to compare differences in effects of different glucocorticoids on expression of glucocorticoid receptor and regulation of NF-κB/IκB in human keratinocyte HaCaT cell line, and to investigate effects of TNF-αon expression of glucocorticoid receptor and regulation of NF-κB/IκB in human keratinocyte HaCaT cell line, for further insights into the mechanism of tachyphylaxis and resistance to topical glucocorticoid therapy.Methods and Results1. The expressions of GRαin chronic dermatitides (chronic eczema and lichen simplex chronicus) and control normal skin specimens were analyzed using Streptavidin peroxidase (SP) immunohistochemical technique. Immunohistochemistry analysis reconfirmed that expression of GRαwas down-regulated in chronic dermatitides compared with control normal skin.2. Cultured HaCaT cells were treated by dexamethasone with different concentrations and for different durations. The expressions of GRα,βand IκB-αmRNA and protein were examined by Reverse transcription-Real time polymerase chain reaction (RT-PCR) and Western blotting. The expressions of NF-κB neucleoprotein were examined by Western blotting. Dexamethasone could down-regulate the expression of GRαin HaCaT cells in a time-dependent manner, while dexamethasone could up-regulatewith the expression of GRβin HaCaT cells in a time- and dose-dependent manner. Dexamethasone could up-regulate the expression of IκB-αmeanwhile down-regulate with the expression of NF-κB neucleoprotein in HaCaT cells in a dose-dependent manner. The effects were most obvious with treatment of dexamethasone for 36 h.3. Cultured HaCaT cells were divided into hydrocortisone stimulation group and dexamethasone stimulation group and betamethasone stimulation group. The expressions of GRα,βand IκBαmRNA and protein were examined by Reverse transcription-Real time polymerase chain reaction (RT-PCR) and Western blotting. The expressions of NF-κB neucleoprotein were examined by Western blotting. The three kinds of the glucocorticoids with different efficacy could down-regulate the expression of GRαin HaCaT cells, hydrocortisone had most poorly effect while no difference between dexamethasone with betamethasone exists. The three kinds of glucocorticoid could up-regulate the expression of GRβin HaCaT cells,but no difference exists among them. These glucocorticoid could up-regulate the expression of IκBαmeanwhile down-regulate the expression of NF-κB neucleoprotein in HaCaT cells, hydrocortisone had most poorly effect whil no difference between dexamethasone with betamethasone.4. Cultured HaCaT cells were treated by TNF-αwith different concentrations and for different durations. The expressions of GRα,βand IκBαmRNA and protein were examined by Reverse transcription-Real time polymerase chain reaction (RT-PCR) and Western blotting. The expressions of NF-κB neucleoprotein were examined by Western blotting. TNF-αhad no effects on expression of GRαin HaCaT cells, but up-regulate the expression of GRβin HaCaT cells in a time- and dose-dependent manner. TNF-αcould down-regulate the expression of IκBαmeanwhile up-regulate the expression of NF-κB neucleoprotein in HaCaT cells in a dose-dependent manner. The effects were most obvious with treatment of TNF-αfor 1h then went down.Conclusion1. Decreased expression of GRαin chronic dermatitides might interfere with anti-inflammation mechanism in cutaneous lesions and limit therapeutic effect of topical glucocorticoid agents.2. The different regulations on the expression of GRαand GRβin keratinocytes by GC may partly explain the tachyphylaxis and resistance to topical glucocorticoid therapy. Glucocorticoid might suppresse inflammatory reaction by affecting NF-κB/IκB in skin lesions.3. The different effects on the expression of GRαand GRβin keratinocytes by different GC could give guidance for correctly using glucocorticoids with different efficacy. The different effects on the NF-κB/IκB in keratinocytes by different GCs could be helpful to control different inflammatory situations in dermatitides. 4. TNF-αcan increase the expression of GRβ, which may be one of the mechanisms of glucocorticoid resistance induced by inflammatory cytokines. TNF-αmight participates in inflammatory process via regulating NF-κB/IκB in keratinocytes.更多还原