【作者】 阎衡；

【导师】 郝飞；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 一、研究背景和目的银屑病(psoriasis)是一种常见易复发的慢性炎症性皮肤病,对患者的身心健康影响甚大。长期以来银屑病是皮肤病领域研究的重点和热点,较为公认的是T细胞介导的自身免疫反应性炎症机制,但一些临床现象却不能完全用T细胞诱导的免疫损伤来解释。银屑病最常见的是寻常型,其特征性病理改变为Munro微脓肿,表现为皮肤角质层内中性粒细胞(polymorphonuclear neutrophils,PMNs)聚集。Munro微脓肿仅存在于角化不全区,该区角质形成细胞(keratinocyte,KC)有丝分裂率明显增高。从临床角度看,寻常型银屑病同一块皮损可分为“慢性”和“急性”区,“慢性”区镜下为真皮内T细胞大量浸润并向表皮趋化移行,“急性”区镜下则可见Munro微脓肿形成。“慢性”区恢复较快,而“急性”区对治疗抵抗,恢复较慢。这种临床和组织学上具有的“异质性”特点提示PMNs在寻常型银屑病发病中必然起着某种作用。而脓疱型银屑病的病理表现为海绵状脓疱(Kogoj脓肿),疱内大量PMNs浸润,此处KC的增殖水平甚至高于寻常型银屑病。不同条件下,PMNs在银屑病发病中的作用及其机制仍缺乏深入研究。虽有学者提出以PMNs为主的非感染性炎症反应仍由T细胞介导,鉴于以上现象,PMNs在其中所扮演的角色仍不容忽视。PMNs是非特异性炎症反应的主要效应细胞之一,近年来发现不仅PMNs产生的各类蛋白酶具有免疫调节作用,而且能够合成、分泌高活性的细胞因子。中性粒细胞弹性蛋白酶(neutrophil elastase,NE)是PMNs研究的热点和焦点。NE属于丝氨酸蛋白酶的糜蛋白酶超家族成员,具有防御、组织损伤、促分泌、炎症放大等作用,已证实在急性肺损伤等疾病中起着关键作用,并由此提出的NE与内源性蛋白酶抑制剂平衡失调的致病学说已得到广泛认同。NE通过EGFR信号通路可刺激KC增殖,部分解释了寻常型银屑病具有PMNs的皮损部位增殖加快的现象。NE还具有前炎症因子效应,可诱导多种细胞因子的表达,形成复杂的细胞因子网络,产生“级联瀑布”效应,放大炎症反应。NE的内源性抑制剂trappin-2蛋白主要由角质形成细胞及上皮细胞分泌,通过降解NE有效地抑制NE活性。trappin-2在正常皮肤不表达,而表达于炎症性皮肤病及某些皮肤肿瘤。已证实trappin-2可以促进KC及肿瘤细胞的分化,是KC及某些肿瘤分化的标志蛋白。Trappin-2通过有效抑制NE活性,间接抑制细胞增殖,同时还能够直接抑制某些肿瘤细胞的增殖。Trappin-2在肺损伤等疾病中发挥抗炎作用,能够减少IL-6、IL-8等细胞因子的释放,可以作为抑制过度炎症反应的有效手段。由此可见,NE和trappin-2间的平衡对增殖和炎症反应具有重要的作用。目前关于NE和trappin-2在银屑病中的作用及其与银屑病发生发展的关系尚未见系统的研究报道。因此我们拟通过研究NE和trappin-2在银屑病中的表达,以及两者在银屑病增殖及炎症异常中的调控作用,探讨NE和trappin-2在银屑病发病过程中的作用机制,从而部分阐明银屑病的发病机制,研究结果还对寻求银屑病新的治疗靶位和方法具有一定的指导意义。二、方法与结果1.通过酶联免疫吸附试验(ELISA)对NE和trappin-2在寻常型银屑病和脓疱型银屑病血清及皮损组织液中的表达水平进行定量研究。结果表明银屑病血清及皮损中NE和trappin-2水平均明显高于健康对照组(P<0.01),其中寻常型银屑病血清中NE和trappin-2水平与病情活动相关,Pearson相关系数分别等于0.632、0.541,P值均<0.01。虽然脓疱型银屑病中NE水平显著高于寻常型银屑病组,trappin-2表达水平却并未相应增高,NE与trappin-2的表达水平间存在失衡现象。2.为了证实NE和trappin-2参与了银屑病的增殖调控,我们构建了trappin-2真核表达载体pIRES2-EGFP-trappin-2,用脂质体法转染人HeLa细胞,获得trappin-2高表达上清(97ng/ml),再将NE和trappin-2高表达上清作用于HaCaT细胞和银屑病跨膜模型。MTT和3H-TdR掺入试验结果均表明NE可以明显促进HaCaT细胞的增殖(P<0.01),在一定浓度范围内随着NE浓度增高,其促增殖作用逐渐增强。NE的强效抑制剂西维来司钠(sivelestat)可有效阻断NE的促增殖作用。免疫组化染色证实10 IU/L NE可增强银屑病跨膜模型Ki67和p53表达水平(P<0.01或P<0.05)。表明NE在银屑病中具有明显的促增殖作用。另外,高表达trappin-2上清作用于HaCaT细胞后,MTT和3H-TdR掺入试验的变化率与对照组间差异显著(P<0.01),并呈剂量依赖关系。97ng/ml trappin-2可有效减弱银屑病皮损Ki67和p53的表达水平(P<0.01)。提示trappin-2具有抑制银屑病增殖的作用。3.采用ELISA法检测上清中IL-6、IL-8表达水平,免疫印迹(Western-blot)法检测HaCaT细胞和银屑病患者外周血单一核细胞(peripheral blood mononuclear cells,PBMC)中ICAM-1的表达,通过免疫组化染色检测银屑病跨膜模型的ICAM-1表达水平。结果表明:NE并不能单独增强HaCaT细胞分泌IL-6、IL-8和表达ICAM-1水平,但可以显著增加TNF-α刺激下三类因子表达水平(P<0.01)。NE还可以促进银屑病PBMC和皮损的炎症因子表达水平(P<0.01)。因此,NE在银屑病中可以作为炎症增强因子,推动银屑病的炎症进程。另外,高表达trappin-2上清与TNF-α协同作用于HaCaT细胞,则能有效减弱IL-6、IL-8分泌及ICAM-1表达的水平(P<0.05)。高表达trappin-2上清单独作用于银屑病PBMC和皮损同样可以减弱三类因子表达水平(P<0.01或P<0.05)。因此,trappin-2在银屑病中可能作为炎症抑制因子,减缓银屑病的炎症进程。三、结论1.NE和trappin-2在银屑病血清和皮损组织液中的表达均明显高于健康对照组,部分表达水平和疾病活动性正相关,并且脓疱型银屑病中NE与trappin-2表达水平间存在失衡现象。2.将NE和trappin-2高表达上清作用于HaCaT细胞和银屑病跨膜模型,发现NE具有明显的促增殖作用,而trappin-2具有一定的抑制增殖作用。3.NE并不是典型的炎症因子,但作为一种炎症增强因子可以增加银屑病中IL-6、IL-8及ICAM-1表达水平。Trappin-2可能通过降低这些细胞因子的表达水平,来减弱银屑病中的炎症反应程度,有利于疾病恢复。通过以上实验,为评价NE/trappin-2在银屑病发病机制中的作用和地位,寻求新的治疗靶点奠定了一定的实验基础。更多还原

【Abstract】 Background and objectivesPsoriasis is a common, recurrent, chronic inflammatory skin disease, which greatly influences the physical and mental health of patients. Therefore, psoriasis has long been a focus of research in dermatology. T lymphocyte-mediated autoimmune reaction is considered as a putative mechanism of this disease. However, this mechanism cannot thoroughly explain clinical findings of psoriasis.Psoriasis vulgaris is the commonest type of psoriasis, which is pathologically characterized by Munro microabscess in skin lesions, i.e., aggregation of polymorphonuclear neutrophils(PMNs) in the stratum corneum. Munro microabscess exists only in parakeratosis areas in which the mitosis rate of keratinocytes increases significantly. Clinically, there are both“chronic”and“acute”areas in the same skin lesion of psoriasis vulgaris. In the“chronic”region, infiltration of large numbers of T lymphocytes in the dermis and migration of these cells toward the epidermis are observed microscopically. In the“acute”area, Munro microabscess is observed microscopically. The“chronic”area recovers quickly, while the“acute”area is refractory to treatment and recovers slowly. Such a clinical and histological“heterogeneity”suggests PMNs play a role in pathogenesis of psoriasis vulgaris. In psoriasis pustulosa, there are spongiform pustules (Kogoj abscess) with infiltration of many PMNs. The keratinocyte proliferation level is higher in psoriasis pustulosa than in psoriasis vulgaris. The role of PMNs in the pathogenesis of psoriasis has not been investigated thoroughly. Although, it was suggested that non-infective inflammatory reaction dominated by PMNs is mediated by T lymphocytes, the role of PMNs in the pathogenesis of psoriasis also should be further investigated.PMNs are one of the major effector cell types involved in non-specific inflammatory reactions. Over recent years, PMNs have been found to produce various immunoregulating proteases and synthesize and secrete highly active cytokines. Of these immunoregulating proteases, neutrophil elastase (NE) is a focus of interest. As a member of the serine protease/chymotrypsin superfamily, NE plays a role in tissue injury, secretion promotion, and augmentation of inflammation. It has been shown that NE plays a key role in such diseases as acute lung injury. An imbalance between NE and endogenous protease inhibitors is widely recognized as a mechanism of these diseases. NE stimulates keratinocyte proliferation through the EGFR signal pathway, which partially explains the accelerated proliferation of skin lesions with PMNs infiltration in psoriasis vulgaris. NE also acts as a proinflammatory factor, inducing the expression of multiple cytokines, forming complex cytokine networks, and resulting in cascade reactions and augmentation of inflammatory reactions. Trappin-2 protein is an endogenous NE inhibitor mainly secreted by keratinocytes and epithelial cells, which can effectively suppress NE activity through degrading NE. Trappin-2 is not expressed in the normal skin, but in inflammatory skin diseases and certain skin tumors. Trappin-2 has been shown to promote the differentiation of keratinocytes and tumor cells, and can be used as a differentiation marker for keratinocytes and certain tumor cells. Trappin-2 suppresses cell proliferation through suppressing the NE activity, and also directly suppresses the proliferation of certain tumor cells. Trappin-2 plays an anti-inflammatory role in such diseases as lung injury, and reduces the release of cytokines such as IL-6 and IL-8. Hence, trappin-2 can be utilized to suppress excessive inflammatory reactions. The balance between NE and trappin-2 is crucial for cell proliferation and inflammatory reaction.No systematic research on the role of NE and trappin-2 in psoriasis and the relationship between NE and trappin-2 and the pathogenesis and severity of psoriasis has been reported up to now. Hence, we investigated the expression of NE and trappin-2 in psoriasis, and the role of NE and trappin-2 in the psoriasis proliferation and inflammation, so as to elucidate the pathogenesis of psoriasis and find new therapeutic targets and methods of psoriasis.Methods and results1.The expression levels of NE and trappin-2 in serum and skin lesions of patients with psoriasis vulgaris and psoriasis pustulosa were quantitatively analyzed by enzyme linked immunosorbent assay (ELISA). The results demonstrated that both the NE and trappin-2 levels in the serum and skin lesions were significantly higher in the psoriasis group than in the control group (P<0.01), and the serum levels of NE and trappin-2 were positively associated with the activity of psoriasis vulgaris (Pearson correlation coefficient=0.632, 0.541, P<0.01). The NE level was significantly higher in the psoriasis pustulosa group than the psoriasis vulgaris group. But the trappin-2 level did not increase correspondingly in the psoriasis pustulosa group, suggesting an imbalance between NE and trappin-2 expression levels in the psoriasis pustulosa group.2.In order to test if NE and trappin-2 participate in the regulation of psoriasis proliferation, we constructed the trappin-2 eukaryotic expression vector pIRES2-EGFP- trappin-2, and transfected human HeLa cells with the vector using liposome to obtain trappin-2 overexpressed supernatants (97ng/ml). Then NE and trappin-2 overexpressed supernatants were used to treat HaCaT cells and the transwell psoriasis organ culture model. MTT and 3H-TdR incorporation assay demonstrated that NE significantly promoted HaCaT cell proliferation (P<0.01) in a dose-dependent manner within a certain range of NE concentration. Sivelestat, a potent NE inhibitor, effectively blocked the proliferation-promoting effect of NE. Immunohistochemical staining demonstrated that 10 IU/L NE elevated the Ki67 and p53 expression levels in the transwell psoriasis organ culture model (P<0.01, P<0.05), suggesting the significant proliferation-promoting effect of NE in psoriasis.In addition, MTT and 3H-TdR incorporation assay indicated significant, dose-dependent differences between HaCaT cells treated with trappin-2 overexpressed supernatants and the control group (P<0.01). 97ng/ml trappin-2 effectively lowered the Ki67 and p53 expression levels in psoriasis lesions (P<0.01), suggesting psoriasis proliferation suppression by trappin-2.3.The IL-6 and IL-8 expression levels in the culture supernatants were determined by ELISA, ICAM-1 expression on HaCaT cells and PBMCs of psoriasis patients was detected by Western blotting, and ICAM-1 expression in the transwell psoriasis organ culture model was detected immunohistochemically. The results demonstrated that NE alone did not enhance IL-6 and IL-8 secretion or ICAM-1 expression by HaCaT cells, but significantly increased the expression levels of IL-6, IL-8 and ICAM-1 under the stimulation of TNF-α(P<0.01). NE also elevated the expression levels of these inflammatory factors both on PBMCs and in skin lesions of psoriasis patients (P<0.01). Hence, NE may act as an inflammation-enhancing factor in psoriasis and contribute the inflammatory progress of psoriasis.In addition, trappin-2 overexpressed supernatants and TNF-αtogether effectively reduced IL-6 and IL-8 secretion and ICAM-1 expression by HaCaT cells (P<0.05). Trappin-2 overexpressed supernatants alone lowered the expression levels of IL-6, IL-8 and ICAM-1 on PBMCs and in skin lesions of psoriasis patients (P<0.01 or P<0.05). Thus, trappin-2 may act as an inflammatory suppressing factor in psoriasis and slow down the inflammatory process of psoriasis.Conclusions1.The NE and trappin-2 expression levels of serum and skin lesion of psoriasis patients are significantly higher than those in the control group, and partially correlated positively with the activity of psoriasis. Moreover, there is an imbalance between NE and trappin-2 expression levels in psoriasis pustulosa.2.Treatment of HaCaT cells and the transwell psoriasis organ culture model with NE and trappin-2 overexpressed supernatants indicates significant proliferation-promoting effect of NE and certain proliferation suppressing effect of trappin-2.3.NE is not a typical inflammatory factor. But as an inflammation-enhancing factor, it increases the expression levels of IL-6, IL-8 and ICAM-1 in psoriasis. Trappin-2 attenuates inflammatory reactions through lowering the expression levels of IL-6, IL-8 and ICAM-1 in psoriasis, thus helping recovery of the disease.This study provides an experimental basis for evaluating the role of NE/trappin-2 in the pathogenesis of psoriasis and for finding new therapeutic targets.更多还原