【作者】 周炯；

【导师】 郑敏；

【作者基本信息】 浙江大学 ， 内科学， 2012， 博士

【摘要】 研究背景在过去的20年中,皮肤中整合素和ECM的表达成为了研究热点。整合素家族和ECM蛋白在皮肤的不同区域和发育不同时期的表达变化造成了不同的粘附环境,使皮肤能够更好地利用不同的细胞。这对于维持表皮结构和功能的稳定是十分有力的。硬皮病和银屑病作为皮肤科的两大常见疾病,与整合素以及ECM的关系也早有研究。然而,有研究者指出银屑病病变发生在皮肤全层,而不是仅有表皮的异常。那么是否硬皮病的表皮也牵涉于发病机制之中呢?银屑病是以表皮过度增殖、血管内皮异常增生和淋巴细胞浸润为特征的慢性炎症性皮肤病。而纤维化、血管损伤甚至栓塞是硬皮病患者后期的主要病理特征。根据银屑病和硬皮病的两极化病理表现,我们大胆假设,在银屑病和硬皮病患者的角质形成细胞和真皮成纤维细胞中可能存在截然相反的分子改变,需要进一步大量的实验数据支持这一假说。因此,我们进行了相关的研究。目的明确整合素α5和β4在正常人表皮,硬皮病和银屑病皮损区角质形成细胞中的表达情况,重点研究不同生理、病理状态下的角质形成细胞中不同细胞因子对整合素α5和β4的调节作用；某些调控因素如Ca++及地塞米松对正常皮肤,硬皮病/银屑病皮损成纤维细胞ECM蛋白,包括FN, CTGF, col-1A的表达的影响,及可能涉及的细胞通路。方法第一部分：活检皮肤一分为二,一部分包埋制成蜡块。另一部分分离、培养角质形成细胞,提取总蛋白质。以免疫组化法检测整合素α5和β4在正常皮肤,硬皮病/银屑病皮损的表达情况。以WB法检测正常/硬皮病/银屑病表皮角质形成细胞中整合素β5和/β4蛋白质的表达水平；使用外源性IL-1β,IL-13,IL-17A,TNF-α, TGF-β1, ET-1, IFN-γ,VEGF165作为刺激因子,观察它们对不同生理、病理状态下的表皮角质形成细胞整合素a5和ββ4蛋白质的调节。第二部分：分离、培养正常真皮及硬皮病、银屑病皮损区成纤维细胞,提取总蛋白质。以WB法检测Ca++和地塞米松对成纤维细胞ECM蛋白质(FN, CTGF,col-1A)表达的影响；以ERK/Rho/p53抑制剂阻断相应细胞通路,了解影响成纤维细胞ECM蛋白质表达的可能机制。结果1、整合素α5和β4在正常皮肤,硬皮病/银屑病皮损中的定位和表达1)整合素α5和ββ4在硬皮病中的表达：α5在正常人和硬皮病患者表皮基底层和邻近基底层的棘细胞层表达较强,颗粒层和邻近颗粒层的棘细胞层表达较少,角质层没有表达；而β4表达于正常人和硬皮病患者表皮各层,但是在基底层表达极弱。然而β4在正常人表皮细胞的胞膜和胞浆均有表达,在硬皮病患者表皮仅在胞膜表达。a5在硬皮病患者的角质形成细胞表达下降,但是无显著性差异；而β4在硬皮病患者的角质形成细胞表达下降,且有显著性差异。2)整合素α5和β4在银屑病中的表达：α5在正常人表皮除角质层外的各层具有表达,基底层和邻近基底层的棘细胞层表达较强；而在银屑病表皮的基底层和棘细胞层均匀表达,为强阳性。β4表达于正常人表皮各层,但是在基底层表达极弱；然而在银屑病患者表皮的角质层和基底层无表达。α5和β4在正常人和银屑病患者的表皮角质形成细胞中的蛋白表达量亦有所差异：α5在银屑病患者的角质形成细胞表达增加,且有显著性差异；而β4在银屑病患者的角质形成细胞表达下降,但是无显著性差异。3) IL-17A、ET-1、TNF-α、IFN-γ对正常人和硬皮病患者表皮角质形成细胞中整合素α5和β4的调节作用：正常人表皮角质形成细胞中, IL-17A作用下,α5蛋白质表达略有增加；ET-1作用下,α5表达变化不明显；而TNF-α或IFN-γ处理后,a5蛋白质表达增加。而在硬皮病表皮角质形成细胞中,IL-17A作用使α5蛋白质表达增加；ET-1、TNF-α或IFN-γ作用后,α5蛋白质表达显著增加。正常人表皮角质形成细胞中,IL-17A、ET-1、TNF-α或IFN-γ作用后,β4蛋白质表达均增加。而在硬皮病表皮角质形成细胞中,IL-17A、ET-1、TNF-α或IFN-γ作用后,β4蛋白质表达基本无变化。4) IL-13、IL-17A、ET-1、TNF-α、IFN-γ、IL-1β、TGF-β1、VEGF165对正常人和银屑病患者表皮角质形成细胞中整合素α5和β4的调节作用：正常人表皮角质形成细胞中,IL-13、ET-1、IL-17A、TNF-α或IFN-γ作用后,a5蛋白质表达增加,TGF-β1、VEGF165对a5无影响,而IL-1β使α5表达下降。而IL-13、IL-17A、ET-1、TNF-α、IFN-γ、IL-1β、TGF-β1或VEGF165处理后,β4蛋白质表达均增加,尤其是IL-13、IFN-γ、IL-1β、TGF-β1和VEGF165。然而,在银屑病表皮角质形成细胞中,IL-13、ET-1、TNF-α、 IFN-γ、VEGF165处理,能使α5表达减少；IL-17A能使其明显减少；IL-lβ或TGF-β1对α5无影响。IL-17A或TNF-α作用24h后,β4蛋白质表达减少；而IL-13、ET-I、IFN-γ、IL-1β、TGF-β1和VEGF165作用后,β4蛋白质表达无影响。5) IL-13、IL-17A、ET-1、TNF-α、、IFN-γ通过ERK通路调节正常人和银屑病表皮角质形成细胞的整合素a5和β4的表达：U0126能够减少正常人表皮角质形成细胞的整合素a5的蛋白表达,而对β4的表达无影响。相反地,在银屑病角质形成细胞中,U0126能够明显减少整合素β4的蛋白表达,而对α5的表达影响不大。U0126预处理的正常人表皮角质形成细胞中,IL-13、ET-1能够使α5表达增加；IL-17A、TNF-α和IFN-γ能够显著增加玟5的表达。而IL-13、IL-17A和IFN-γ会减少β4的蛋白表达：但是ET-1和TNF-α能够增加其表达。U0126预处理的银屑病患者表皮角质形成细胞中,IL-13、IL-17A能够显著增加α5表达；而IFN-γ能够减少其表达；ET-1和TNF-α对a5表达无影响。IL-13、IL-17A可以增加β4的蛋白表达；ET-1、TNF-α和IFN-γ对该蛋白的表达无影响。2、钙离子和地塞米松对正常皮肤及硬皮病、银屑病皮损成纤维细胞中FN,CTGF,col-1A的调节作用1)在正常成纤维细胞中,Ca++对FN和CTGF都有很强的促进作用,对col-1A的影响不大。而在硬皮病和银屑病的成纤维细胞中,Ca++对FN,CTGF和col-1A均有促进作用。2)证明ERK,Rho,p53和Smad2信号通路都与Ca++对成纤维细胞的影响相关。3)在dex实验中发现,正常成纤维细胞中,dex对FN和col-1A均有抑制作用；但是对CTGF却有促进作用。而在硬皮病和银屑病成纤维细胞,dex对上述三种ECM蛋白均有诱导产生的作用。4)证明ERK,Rho,p53和Smad2信号通路都与dex对成纤维细胞的影响相关。结论1、整合素α5在正常人和硬皮病患者表皮除角质层外的各层具有表达,基底层和邻近基底层的棘细胞层(暂时扩增细胞)表达较强,而在银屑病表皮的基底层和棘细胞层均匀表达,为强阳性；2、整合素β4表达于正常人和硬皮病患者表皮各层,在基底层表达极弱。β4在正常人表皮细胞的胞膜和胞浆均有表达,在硬皮病患者表皮仅在胞膜表达。银屑病患者表皮的角质层和基底层不表达β4；3、体外培养的表皮角质形成细胞a5蛋白质表达：和正常角质形成细胞相比,硬皮病皮损角质形成细胞中α5表达下降,但无显著性差异。而在银屑病皮损角质形成细胞中表达明显增高,且有显著性差异；4、体外培养的表皮角质形成细胞β4蛋白质表达：和正常角质形成细胞相比,硬皮病皮损角质形成细胞β4表达明显减少,有显著性差异。而在银屑病皮损角质形成细胞中表达虽然下降,但无显著性差异；5、接受同种细胞因子刺激后,正常皮肤和硬皮病、银屑病皮损角质形成细胞的整合素α5和β4调节各不相同；6、钙离子可能在硬皮病和银屑病的发病中发挥重要作用；7、地塞米松对硬皮病和银屑病的成纤维细胞的细胞外基质蛋白产生有促进作用。更多还原

【Abstract】 BACKGROUNDIn the past20years, integrin family and ECM component become the hot-spot in research field of dermatology. Expression of the two groups of molecules is variable during the different period in skin development, and in different parts of skin, in order to make the different adhesion environment, to utilize the different cells better. It is very important for maintaining the epidermal structure and stable function. Scleroderma and psoriasis are two common skin diseases, and their relation with integrin and ECM have been studied for years. However, some researchers figured out that the entire skin is abnormal in psoriasis patients, not only in epidermis. Have the epidermis also involved in mechanism of scleroderma?Psoriasis is a chronic skin inflammation characterized with epidermal hyper-proliferation, abnormal proliferation of vascular and lymphocyte infiltration. Fibrosis and vascular impairment are the main pathological characteristics of scleroderma. Considering the opposite manifestations of the two diseases, we assume that there may be reversed molecular alteration in keratinocytes and fibroblasts between scleroderma and psoriasis.OBJECTIVESTo investigate the expression of integrin α5,β4in normal, scleroderma and psoriatic epidermal keratinocytes, the regulation of integrin α5,β4by cytokines in keratinocytes under different status; the regulation of ECM component by Ca++and dexamethasone in normal, scleroderma and psoriatic fibroblasts and the probable cell signal pathways involved.METHODSIn the first part:skin biopsies were divided into two parts, one part was paraffin embedded, and the other part was used to culture keratinocytes. The total protein was extracted from cultured epidermal keratinocytes. The protein level of integrin α5,β4was determined by western blot. We further localized the integrin α5,β4in normal, scleroderma and psoriatic epidermis by immunohistological staining. The expression of integrin α5,β4was observed by western blot after adding exogenous IL-1β,IL-13, IL-17A, TNF-α, TGF-β1, ET-1, IFN-γ and VEGF165.In the second part:the culture fibroblast from normal, scleroderma and psoriatic dermis were treated with Ca++and dexamethasone at the presence or absence of U0126, Y27632, pifithrin-α. The total protein was extracted from cultured dermal fibroblasts, and the protein was determined by western blot to investigate the expression of fibronectin, CTGF and type I collagen. RESULTS1、Localization and expression of integrin α5,β4in normal, scleroderma and psoriatic epidermis:1) Integrin a5localized in the whole epidermis except for squamous layer both in normal and scleroderma epidermis. In normal and scleroderma epidermis, the distribution of a5was both distinguished in the basal layer. Integrin β4was distributed in the whole epidermis, but faint in the basal keratinocytes, both in normal and scleroderma epidermis. Furthermore, the cellular distribution of β4in normal keratinocytes was membranous and cytoplasmic, while predominantly membranous in scleroderma keratinocytes. Western blot demonstrated that integrin α5and β4was decreased in scleroderma keratinocytes, comparing with normal keratinocytes;2) α5localized in the whole skin, except for squamous layer in psoriatic epidermis. In normal epidermis, the expression of α5was very weak, but showed a relative concentration at the basal layer. In psoriasis, the distribution of α5was almost homogenous in basal and prickle keratinocytes, and the immunoreaction was much stronger than that in normal epidermis. Integrin β4was detected in the membrane of normal epidermal keratinocytes from basal to granular layers, while β4being absent in the basal and parakeratosis layer of psoriatic epidermis. Furthermore, expression of α5in normal keratinocytes was less than that in psoriatic keratinocytes significantly, while no significant difference of β4being observed;3) In normal keratinocytes, integrin a5was increased by TNF-α and IFN-γ, but not altered by IL-17A and ET-1. However, in scleroderma keratinocytes, integrin α5was upregulated by IL-17A, especially significantly by ET-1, TNF-α, and IFN-γ. That is, scleroderma keratinocytes response more sensitive to IL-17A, ET-1, TNF-α and IFN-y than in normal keratinocytes, in production of integrin α5. Integrin β4in normal keratinocytes was increased by IL-17A, ET-1, TNF-α and IFN-γ, while in scleroderma keratinocytes, no obvious difference was observed of β4between IL-17A-, TNF-α-, IFN-γ-, ET-1-treated samples and controls;4) In normal keratinocytes, α5was upregulated by IL-13, ET-1, especially by IL-17A, TNF-α and IFN-γ, not altered by TGF-β1and VEGF165, but decreased by IL-1β.β4was increased by IL-17A, ET-1, TNF-α, particularly by IL-13, IFN-y, IL-1β, TGF-β1and VEGF165in normal keratinocytes. However, in psoriatic keratinocytes, α5was decreased by IL-13, ET-1, TNF-α, IFN-γ, VEGF165, especially IL-17A, not being altered by IL-1/3and TGF-β1.β4was decreased by IL-17A and TNF-α, not being altered by IL-13, ET-1, IFN-γ, IL-1β, TGF-β1and VEGF155;5) In normal keratinocytes,10μM U0126prominently decreased a5, but not alter the expression of β4. On the contrary, in psoriatic keratinocytes, integrin α5was nearly not altered, but β4was decreased very obviously, by10,uM U0126. In normal keratinocytes, in the presence of U0126, α5was increased by IL-13, ET-1and especially, IL-17A, TNF-a and IFN-γ.β4was decreased by IL-13, IL-17A, and IFN-γ, but being increased by ET-1and TNF-α. In psoriatic keratinocytes, with addition of U0126, a5was strongly increased by IL-13and IL-17A, not being altered by ET-1and TNF-α, but being decreased by IFN-γ. β4was increased by IL-13and IL-17A, not being altered by ET-1, TNF-α and IFN-γ.2、Regulation of FN, CTGF and col-1A by Ca++and dexamethasone1) In normal fibroblasts, Ca++induced enhancement of FN and CTGF, and have no effect on col-1A production. However, in both scleroderma and psoriatic fibroblasts, Ca++increased FN, CTGF and col-1A production.2) ERK, Rho, p53and Smad2signal pathways were involved in the regulation of ECM component in fibroblasts by Ca++3) In normal fibroblasts, dexamethasone shows suppression of FN and co-1A production, but enhancement of CTGF. However, in both scleroderma and psoriatic fibroblasts, dexamethasone increased FN, CTGF and col-1A production.4) ERK, Rho, p53and Smad2signal pathways were involved in the regulation of ECM component in fibroblasts by dexamethasone.Conclusion1. Integrin α5was distributed diffusely in normal and scleroderma epidermis, and expressed strong immune-activity in basal layer and the adjacent prickle layer; and α5was strong positive and homogenous in basal and prickle layer in psoriatic epidermis;2.β4was distributed in normal and scleroderma epidermis diffusely, but weakly in basal layer.β4was expressed in cell membrane and cytoplasm in normal keratinocyte, but only in cell membrane in scleroderma keratinocyte. There was no β4expression in stratum corneum and basal layer in psoriatic epidermis;3、Comparing with normal keratinocyte, integrin α5expression was decreased in scleroderma keratinocyte, but increased in psoriatic keratinocyte significantly; 4、Comparing with normal keratinocyte, integrin β4expression was decreased in scleroderma keratinocyte significantly, and decreased in psoriatic keratinocyte. but not significantly;5、With the same stimulation, the regulation of integrin α5and β4in normal, scleroderma and psoriatic keratinocytes was different;6、Calcium may play an important role in etiology of scleroderma and psoriasis;7、Dexamethasone will induce ECM protein promotion in scleroderma and psoriatic fibroblasts.更多还原