【作者】 颜薇；

【导师】 赵振民；

【作者基本信息】 北京协和医学院 ， 外科学， 2011， 博士

【摘要】 研究背景：我们通常将皮肤老化分为内源性老化与外源性老化两类。影响后者最重要的因素就是紫外线UVA和UVB的照射,由此导致的皮肤老化即光老化。紫外线辐射早期仅引起皮肤皱纹,萎缩,色素沉着等老化表现,后期严重时则可诱发相应的皮肤疾病,甚至皮肤肿瘤。虽然目前对于光老化的研究很多,但对其生物学机制的了解尚不够深入。同时,目前对于皮肤光老化研究仍主要集中于皮肤单种细胞或皮肤亚单位(真皮,表皮或结缔组织)研究范围中,少有对全层皮肤整体的研究,尤其对亚裔汉族女性全层皮肤光老化研究甚少。本研究期望以汉族女性全层皮肤作为研究对象在基因水平上研究皮肤光老化的相关基因及生物学机制。研究方法：我们选取来我院做全颜面除皱手术的21名女性患者作为研究对象,所有患者均统一了光老化程度,居住地域,发式,月经史,家族史、既往疾病史等可能存在的影响因素。收集患者手术过程中废弃的耳前皮肤(光暴露区)和耳后皮肤(光保护区)作为配对的光照组和对照组,应用Illumina芯片进行全基因组扫描寻找差异基因。对结果进行相应的统计学分析,并应用G0对基因进行功能聚类分析,应用WebGestalt, DAVID和GSEA软件对基因进行功能富集分析。应用Q-RT-PCR对差异基因进行进一步验证。研究结果：我们共发现1762个差异表达基因(P<0.05)。其中,上调差异表达基因829个,下调差异表达基因933个。通过GO数据库对基因功能注释并进行基因功能聚类分析发现,这些差异表达基因主要参与：免疫应答,黏附与受体相关生物学过程,信号转导,转录调节,炎症反应,酶相关生物学过程,金属离子结合,胶原与纤维蛋白相关生物学过程,细胞凋亡,血管发生,细胞增殖分化和发育,皮肤发育,代谢等生物学过程。同时,我们应用WebGestalt, DAVID和GSEA软件中的" KEGG Table and Maps ", " KEGG Functional Annotation Chart"以及"KEGG pathways gene sets "功能模块进行分析,发现该差异基因共富集通路11个,其中上调通路3个,下调通路8个,包括细胞外基质受体交互作用,TGF-β信号通路,细胞循环,缬氨酸、亮氨酸、异亮氨酸降解,精氨酸、脯氨酸代谢,果糖、甘露糖代谢,鞘糖脂生化合成,色氨酸代谢,花生四烯酸代谢,氮代谢等。选择HOXA5,LEPR, CLDN5,LAMC3以及CGA基因进行Q-RT-PCR验证,结果一致性好。研究结论：我们的研究显示皮肤光老化是一个由多个基因和生物学通路参与的复杂生物学过程。其中有部分结果与以往研究互为验证,但是仍然有很多基因及生物学通路以往未有类似报道。考虑我们是以全层皮肤作为研究对象,能更客观反应了光老化皮肤的生物学网络作用。我们的研究结果将为进一步了解皮肤光老化机制及其相关疾病发生机理和临床治疗提供可信线索。我们仅仅对相关基因和生物学通路做了初步的探索和展示,仍需要更细致和深入的研究来了解其具体的作用机制。研究背景：皮肤老化像一面镜子可以反映人体的整体老化进程。皮肤老化可根据老化因素分为外源性老化和内源性老化。内源性老化主要受遗传背景、机体内在内分泌环境、代谢等影响。雌激素作为一种主要的性激素,对皮肤内源性老化,尤其对女性内源性皮肤老化发挥重要作用。女性绝经后,雌激素分泌水平明显下降,并随着年龄增长而逐年递减。伴随雌激素变化,女性皮肤可出现明显老化包括皮肤萎缩,干燥,皱纹增生,潮红等。既往很多研究发现,雌激素与皮肤内源性老化存在密切联系,同时,雌激素替代治疗也间接证明了雌激素对皮肤老化的作用。但是,雌激素对皮肤内源性老化的具体生物学机制目前尚不明确,尤其对于汉族女性雌激素源性内源性皮肤老化机制缺乏清晰的了解。而且,目前对于雌激素源性皮肤老化研究仍主要集中于皮肤单种细胞或皮肤亚单位(真皮,表皮或结缔组织)研究范围中,少有对全层皮肤整体的研究。本研究以全层皮肤作为研究对象在基因水平上探索雌激素源性皮肤老化的相关基因及生物学机制。方法：我们选取来我院做全颜面除皱手术的13名老年女性患者(年龄范围46-55岁,平均50.7岁),根据其月经情况分为2组：绝经组与未绝经组。所有患者均统一了家族史、既往疾病史等可能存在的影响因素。收集患者手术过程中废弃的耳后皮肤(光保护区),处理成全层皮肤后液氮保存,提取RNA并进一步应用Illumina芯片进行全基因组扫描寻找差异基因。对结果进行相应的统计学分析,并对基因进行功能聚类分析和功能富集分析。应用Q-RT-PCR对差异基因进行进一步验证。结果：我们共发现244个差异表达基因(P<0.05)。其中,上调差异表达基因131个,下调差异表达基因113个。通过G0数据库对基因功能注释并进行基因功能聚类分析发现,这些差异表达基因主要参与：新陈代谢、细胞黏附、金属离子结合、转录、信号转导、细胞凋亡、生物合成、泛素循环、免疫应答、炎症、发育等生物学过程。同时,我们应用WebGestalt工具和KEGG数据库对基因进行功能富集分析发现该差异基因共富集通路11个,包括赖氨酸降解、β丙氨酸代谢、MAPK信号通路、胰岛素信号通路、柠檬酸盐循环通路、RNA降解通路等。结论：我们的研究显示雌激素与皮肤内源性老化存在密切的联系。这是一个由多个基因和生物学通路参与的复杂过程。其中有部分结果与以往研究互为验证,但是仍然有很多基因及生物学通路以往未有类似报道。考虑我们是以皮肤作为研究对象,更客观反应了皮肤内源性老化的生物学网络作用。我们的研究结果将对雌激素源性皮肤老化相关疾病以及雌激素替代治疗的临床研究提供可信线索。我们仅仅对相关基因和生物学通路做了一个初步探索和展示,仍需要更细致和深入的研究来了解其具体的作用机制。更多还原

【Abstract】 Background and Objective Skin aging can be classified into intrinsic aging and extrinsic aging. The latter is mainly influenced by ultraviolet, including UVA and UVB, known as photoaging. Photoaging is first characterized with the alteration in wrinkles, analosis and dyspigmentation, et al, and then the photoaging will develop into severe skin diseases, even skin tumour. Although significant advances in recent years have been made in both the organ culture of skin, little is known in detail about the biological processes and the genes involved in them, especially in Chinese Ham females’skin. Further more, most of these studies are mainly focused on some skin cell (e.g fibroblast, keratinocyte, or melanocytes) or some subunit of skin only (e.g epidermis, dermis, or connective tissue), ignoring the whole biological network based on complete skin tissue. Our study is designed to find out all possible genes and biologic processes involved in skin photoaging based on Chinese Han female full-thickness skin at by microarray technology.Methods 21 healthy Chinese Han women were recruited from Plastic Surgery Hospital of PUMC, Beijing, China. All subjects were similar in photoaging degree, living and climate condition, hair style, and all subjects were healthy without special aging related family history nor smoking and alcoholism history. All skin samples were obtained from the abandoned skin tissue of pre-auricular (UV explosed) and post-auricular (UV protected) during rhytidectomy surgery and were made into full-thickness skin for further research. Illumina beadchips were used for differentially expressed genes analysis. The differentially expressed genes were then analysed in functional clustering with GO database and functional enriched with WebGestalt, DAVID, and GSEA. Q-RT-PCR was applied to further confirmed the chosen genes.Results Identification of the DEGs among these expressed genes then yielded 1762 genes (up-regulated 829 genes, down-regulated 933 genes), which passed the cutoff of P<0.05. All genes were annotated and functional clustered with GO gene database into immune response, cell adhension and receptor related, signal transduction, transcription regulation, inflammation, enzyme related, mental iron binding, collagen and fibrin, cell apoptosis, vasculogenesis, cell proliferation,differentiation, and development, skin development, metabolism, et al. All the genes were functional enriched with analysis tools of WebGestalt, DAVID, GSEA and KEGG database into 11 different pathway, such as ECM-receptor interaction (ECM), TGF-beta signaling pathway (TGF), Cell cycle (CC),Valine, leucine and isoleucine degradation (VLI), Arginine and proline metabolism (APM), Fructose and mannose metabolism (FMM), Glycolysis/ Gluconeogenesis (GG), Glycosphingolipid biosynthesis-ganglioseries (GBG), Tryptophan metabolism (TM), Arachidonic acid metabolism (AAM), Nitrogen metabolism (NM)Lysine degradation, Beta-Alanine metabolism, MAPK signaling pathway, Insulin signaling pathway, Citrate cycle(TCA cycle), mRNA degradation, et al. Five genes, HOXA5,LEPR,CLDN5,LAMC3 and CGA, were chosen for further confirmed by Q-RT-PCR and the Results of the quantitative RT-PCR demonstrated concordance in direction of change between the two different platforms.Conclusion Our study reveals that skin photoaging is a complex biological process with many genes and biological pathway involved in it. Except few genes and pathway reported in the past research, there is still a great difference in results between the present and previous studies. Most of the related genes and biological pathways involved in this field haven’t been reported yet till now. We carried out this study based on full-thickness skin, focusing more on the biological processes network in the whole skin tissue, which provided us more believable and comprehensive information about skin photoaging of Chinese Han female. It will provide us insight information about the effect of skin photoaging and skin photoaging related skin diseases. Our study is a only display of the skin photoaging related genes and pathways. Further study is needed to investigate the biological functions genes and related biological pathway in skin photoaging. Background and Objective The skin is a mirror of the aging process in human. Skin aging can be classified into extrinsic aging and intrinsic aging. The latter is mainly influenced by genetic factor-induced or alterations of the endocrine environment-induced. Estrogen, as one of the sex hormone, plays an important role in intrinsic skin aging, especially in the skin aging of menopausal women. In women, estrogen levels decline rapidly at menopaluse as a result of the loss of ovarian follicles and decrease gradually with the age. This change will accelerate skin aging. It leads to the alternations in skin of dryness, atrophy, fine wrinkling, and hot flashes. Many studies have proved the positive corelation between estragon and intrinsic skin aging directly, and the availability of hormone replacement treatment (HRT) also demonstrates the beneficial effect of estrogen in skin aging indirectly too. Although significant advances in recent years have been made in both the organ culture of skin, little is known in detail about the biological processes and the genes involved in them, especially in Chinese Ham females’skin. Further more, most of these studies are mainly focused on some skin cell (e.g fibroblast, keratinocyte, or melanocytes) or some subunit of skin only (e.g epidermis, dermis, or connective tissue), ignoring the whole biological network based on complete skin tissue. Our study is designed to find out all possible genes and biologic processes involved in estrogen-associated intrinsic skin aging based on Chinese Han female full-thickness skin by microarray technology.Methods 13 healthy Chinese Han women (age ranged 46-55y, average age 50.7y) were recruited from Plastic Surgery Hospital of PUMC, Beijing, China. They were divided into two groups according to their menstrual history. All subjects were healthy without special aging related family history nor smoking and alcoholism history. All skin samples were obtained from the abandoned skin tissue of post-auricular (UV protected) during rhytidectomy surgery and were made into full-thickness skin stored in liquid nitrogen immediately for future research. Illumina beadchips were used for differentially expressed genes analysis. The differentially expressed genes were then analysed in functional clustering and enrichment. Q-RT-PCR was applied to confirmed the chosen genes.Results Identification of the DEGs among these expressed genes then yielded 244 genes (up-regulated 131 genes, down-regulated 113 genes), which passed the cutoff of P<0.05. All 244 genes were annotated and functional clustered with GO gene database into metabolism, cell adhension, mental iron binding, transcription, signal transduction, cell apoptosis, biosynthesis, ubiquitin cycle, immune response, inflammation, development, et al. All the 244 genes were functional enriched with WebGestalt tool and KEGG database into 11 different pathway, such as Lysine degradation, Beta-Alanine metabolism, MAPK signaling pathway, Insulin signaling pathway, Citrate cycle(TCA cycle), mRNA degradation, et al.Conclusion Our study reveals that there is a close correlation between the estrogen and intrinsic skin aging with many genes and biological pathway involved in it. Except few genes and pathway reported in the past research, there is still a great difference in results between the present and previous studies. Most of the related genes and biological pathways involved in this field haven’t been reported yet till now. We carried out this study based on full-thickness skin, focusing more on the biological processes network in the whole skin tissue, which provided us more believable and comprehensive information about estrogen-associated intrinsic skin aging of Chinese Han female. It will provide us insight information about the effect of estrogen in skin aging and estrogen-related skin diseases. Further study is needed to investigate the biological functions genes and related biological pathway in estrogen-associated intrinsic skin aging.更多还原