【作者】 田燕；

【导师】 刘玮；

【作者基本信息】 中国医科大学 ， 皮肤病学与性病学， 2010， 博士

【摘要】 人类长寿基因SIRT1在皮肤光老化进程中的作用研究研究背景皮肤光老化主要是长期紫外线(UV)照射介导的内在损伤的重叠反应。由于长波紫外线(UVA)的穿透能力强,有35%-50%能够到达真皮引起皮肤损伤,即胶原成分的减少和异常弹力纤维沉积,因此长波紫外线被认为是引起皮肤光老化的主要作用波长。皮肤光老化不仅损害人体的容貌而且与皮肤癌有一定关系,因此研究皮肤光老化的发生机制及如何防治光老化早已成为当今研究热点之一。被称为“人类长寿基因”的沉默信息调节因子2(silent information regulator2, Sir2)相关酶类(sirtuins, SIRT)是一种NAD+依赖的去乙酰化酶类,在衰老过程中起着关键的作用,肿瘤抑制因子p53、组蛋白均是其生理性底物。目前的研究认为不同的细胞和不同的环境压力下人类长寿基因SIRT1作用可能不同,因此人类长寿基因SIRT1对细胞寿命的调节作用需要进一步探索。本实验分别从人体和细胞学实验研究人类长寿基因SIRT1在UVA诱导皮肤老化过程中的表达,并通过研究SIRT1对p53活性的影响,对染色体末端端粒(telomere)长度的调控,探讨其在皮肤光老化中的作用机制,从而有助于进一步认识皮肤光老化的发生机理并发现新的抗光老化靶点。由于肿瘤抑制因子p53是SIRT1的生理性底物而且细胞衰老与细胞凋亡、肿瘤发生之间息息相关,因而对于SIRT1基因的表达及其功能的深入研究,不仅可以进一步揭示皮肤衰老的发生机制和本质,而且可以为细胞凋亡和肿瘤的研究提供线索。研究方法本实验包括人体实验和细胞学实验两部分,其中人体实验以10名志愿者的臀部作为受试部位,共分为三个试验区域：阴性对照区域,不进行照射；小剂量区域和高剂量区域分别进行UVA照射,每周照射3次,连续13周,共照射39次。低剂量区域的累积照射剂量为50 J／cm2；高剂量区域的累积照射剂量为1000 J／cm2。照射前后分别测定皮肤角质层含水量、经皮水分流失(Trans epidermal water loss, TEWL)、PH值、反映皮肤颜色的L*a*b值和M、E值。照射前后皮肤活检进行HE染色、弹力纤维和免疫组织化学染色,观察表皮厚度、角质层厚度、胶原纤维、弹力纤维的变化以及与皮肤老化相关的指标——基质金属蛋白酶-1(matrix metallo-proteinase-1,MMP-1)的表达,判定皮肤光老化的存在和程度。免疫组化和Western蛋白印迹法检测SIRT1和p53的表达。细胞学实验采用小剂量亚毒性UVA照射人体皮肤成纤维细胞,连续5次,总照射剂量5 J／cm2。MTT法检测细胞增殖活性,β-半乳糖苷酶染色判定细胞的老化。Western蛋白印迹法检测SIRT1和乙酰化p53的表达；荧光实时定量PCR检测染色末端的端粒长度。在UVA照射的同时加用SIRT1激活剂白藜芦醇和SIRT1抑制剂烟酰胺后,检测乙酰化p53的表达和染色体末端端粒长度的变化。研究结果1.人体皮肤实验结果显示：UVA反复多次照射后角质层含水量下降,经皮水分流失增大, L值下降、M、a、E值升高,PH值无明显变化。皮肤的角质层和表皮厚度增加,真皮中的胶原纤维细碎、淡染,弹力纤维片断化,MMP-1和p53的表达增加,皮肤组织出现光老化的改变。在小剂量组和高剂量组中均出现了上述变化,高剂量组比小剂量组变化的更为明显。2.人类长寿基因SIRT1在正常皮肤组织中有表达。UVA反复多次照射后,皮肤组织中SIRT1的表达增加。在50 J/cm2-1000 J/cm2的照射剂量范围内,剂量越大SIRT1表达量越高。3. SIRT1在人体皮肤成纤维细胞中有表达,亚毒性剂量(5 J/cm2) UVA照射后SIRT1表达增加；毒性剂量(11J/cm2) UVA照射后SIRT1表达减少。4.在UVA照射人体皮肤成纤维细胞的同时加入SIRT1激活剂白藜芦醇,SIRT1的表达量进一步增加,乙酰化p53检测不到。5.UVA照射后皮肤成纤维细胞的端粒长度变短,加入SIRT1激活剂白藜芦醇后可抵消UVA的影响,端粒长度和正常对照组无差别。结论1.UVA反复多次照射可诱导人体皮肤组织发生光老化的改变,皮肤光老化程度与UVA照射剂量相关。2.人类长寿基因SIRT1在UVA诱导的人体皮肤光老化的进程中表达增加,增加程度与照射剂量相关。这种增加可能代表一种针对UVA辐射效应的反馈性机制。3.人类长寿基因SIRT1通过对p53的去乙酰化作用,在皮肤光老化的过程中发挥了保护作用。4.人类长寿基因SIRT1可能通过保护端粒长度,拮抗了UVA引起的皮肤成纤维细胞的衰老。更多还原

【Abstract】 The functions and mechanisms of the longevity gene SIRT1 in the process of human skin pHotoagingSkin pHotoaging is a long term process that results mainly from chronic exposure to solar UVA irradiation. Due to its higher penetration, about 35-50%of UVA reaches to dermis and plays a major role in dermal damage of collagen and elastic fibers. UVA irradiation not only contributes to pHotoaging but also to pHotocarcinogenesis. It has been the focus at present to study the mechanisms, preventions and treatments of skin pHotoaging.SIRT1, which has been called the "human longevity gene" protein, plays a key role in aging process. SIRT1 is a NAD+-dependent deacetylases with both p53 and histones as its pHysiological substrates. Current findings suggest that the effect of SIRT1 may be dependent on both cell type and the context of cellular stress. In our study, the expression of SIRT1 induced by UVA irradiation will be investigated in human skin. To further explore the mechanisms of SIRT1 in skin pHotoaging we asked whether SIRT1 regulates p53 signaling pathway by deacetylation and regulate the length of telomere in vitro. As the close relationship between cellular senescence and carcinogenesis, the further study of SIRT1 expression and effects will be helpful to demonstrate the mechanisms of skin aging and give clues to apoptosis and carcinogenesis.Materials and methodsTwo parts are included in this study. The study in vivo is performed in buttocks of 10 volunteers. Three test sites are as follows:a control site without UVA irradiation; a site with low UVA dose of 50 J/cm2 and a site with high UVA dose of 1000 J/cm2, both exposed to UVA light sources three times a week for 13 weeks. The water of stratum corneum, transepidermal water loss, pH values, the values of L*a*b and M and E are measured pre and post UVA irradiation. Skin samples were obtained after the last time UVA irradiation to study the following parameters as the thickness of stratum corneum and epidermis, changes of collagen and elastic fibers and the expression of MMP-1, SIRT1 and p53 by HE staining, elastic fiber staining and immunohistostaining:The human skin fibroblasts are exposed to low dose UVA once a day for 5 days with total dose of 5 J/cm2. Cellular viability and senescence was evaluated by MTT and SAβ-gal staining, SIRT1 and p53 expression were measured by Western-blot, telomere length by RT-PCR for fibroblasts, which were incubated only or plus resveratrol or nicotinamide.Results1. The results in vivo show:The water of corneum decreased, trans epidermal water loss increases, the values of a*b and M and E increased and L* decreased after UVA irradiation. The thickness of corneum and epidermis increased, collagen and elastic fibers showed morpHologic changes with strong expression of MMP-1.2 The expressions of SIRT1 were found in normal skin. A dose-dependent increase was observed within the range of 50 J/cm2-1000 J/cm2UVA.3. SIRT1 is functionally expressed in cultured skin fibroblasts. The sublethal dose (5 J/cm2) UVA irradiation up-regulates SIRT1 in a dose dependent manner, but the lethal dose (11 J/cm2) UVA down-regulates its expression.4. The acetylation of p53 induced by UVA is abolished by SIRT1 activator resveratrol.5. UVA-induced short telomere length is enhanced by SIRT1 activator resveratrolConclusion1. A dose-dependent skin pHotoaging can be induced by UVA irradiation.2. The expression of SIRT1 increases in the process of skin pHotoaging induced by UVA irradiation. The up-regulated expression of SIRT1 may represent a feedback to UVA irradiation.3. SIRT1 plays a protective role in skin pHotoaging by negative regulation of p53 acetylation.4. SIRT1 functions to inhibit cell senescence may via protection of telomere length.更多还原