应用基因芯片技术探索中国汉族女性皮肤光老化及雌激素源性皮肤固有老化相关基因及作用机制

发布时间：2018-03-16 20:04

本文选题：光老化　切入点：紫外线　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【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(P0.05)。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(P0.05)。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。
[Abstract]:Background: we will usually be divided into endogenous aging skin aging and two types of exogenous factors affecting aging. The latter is the most important UVA and UVB ultraviolet irradiation, resulting in skin aging or photoaging. Ultraviolet radiation early only cause skin wrinkles, pigmentation, atrophy, aging, serious when can cause late the corresponding skin disease, and even skin cancer. Although there is a lot of research for light aging, but its biological mechanism is still not well understood deeply. At the same time, the skin photoaging research is still mainly focused on single skin cells or skin (epidermis or dermis subunit, connective tissue) in the study area, less on the study on the whole layer of the skin, especially the light aging of Asian Chinese women skin little studied. This research is expected to Han female skin as the research object at the gene level. To investigate the related genes and biological mechanisms of photoaging of the skin.
Methods: We selected our hospital 21 women whole face lift surgery as the research object, all patients were unified the degree of light aging, living area, hairstyle, menstrual history, family history, medical history and other factors may exist. The waste collected during surgery in patients with anterior ear skin (light the exposed area) and the posterior auricular skin (light protection zone) as a pair of light group and the control group, the application of Illumina chip for whole genome scans for genes. Appropriate statistical analysis of the results, and the application of G0 on gene function by WebGestalt cluster analysis, DAVID and GSEA on gene function enrichment analysis software the application of Q-RT-PCR. The difference of genes were further validated.
Results: we found 1762 differentially expressed genes (P0.05). Among them, 829 genes up-regulated expression, downregulation of 933 differentially expressed genes. The GO database for annotation of gene function and gene clustering analysis showed that the main parameters and the expression of these genes: differences in immune response and related biological adhesion receptors and process. Signal transduction, transcriptional regulation, inflammatory reaction, enzyme related biological processes, metal ion binding, biological collagen and fibrin, apoptosis, angiogenesis, cell proliferation, differentiation and development, skin development, metabolism and other biological processes. At the same time, we used WebGestalt, DAVID and GSEA in the software of "KEGG Table and Maps", "KEGG Functional Annotation Chart" and "KEGG pathways gene sets" function modules are analyzed, the differences were found for the enrichment pathway 11, up-regulated pathway 3, down 8 pathways, including ECM receptor interaction, TGF- beta signaling pathway, cell cycle, valine, leucine, isoleucine degradation, arginine and proline metabolism, fructose, mannose metabolism, glycosphingolipid biosynthesis, tryptophan metabolism, four arachidonic acid metabolism, nitrogen metabolism. HOXA5 LEPR, CLDN5, Q-RT-PCR, LAMC3 and CGA genes verified, good consistency of results.
Conclusions: our study shows that skin photoaging is an involved by multiple genes and biological pathways of complex biological processes. Some results from previous studies to validate each other, but there are still a lot of genes and biological pathways has never been reported. We are considering to full-thickness skin as the research object, can be more the objective response of photoaging skin biological networks. Our research will further understand the mechanism of skin photoaging and related disease pathogenesis and clinical treatment provide credible clues. We only on related genes and biological pathways to do a preliminary exploration and demonstration, still need more detailed and in-depth study to understand the specific the mechanism of action.
Background: the aging of the skin like a mirror can reflect the whole process of human aging. The aging of the skin can be divided according to the aging factors as exogenous and endogenous aging aging. Endogenous aging is mainly affected by genetic background, the inner body endocrine environment, metabolic effects of estrogen. As a main sex hormone, aging skin endogenous, especially to the skin women play an important role in endogenous aging. After menopause, estrogen secretion decreased significantly, and increase with age and decreased year by year. With the change of estrogen, the female skin can appear obvious aging including skin atrophy, dry wrinkles, hyperplasia, flushing and so on. Many studies have found that the past, estrogen and skin intrinsic aging is closely linked, at the same time. Estrogen replacement therapy also indirectly proved that estrogen on skin aging effect. However, endogenous estrogen on skin aging The biological mechanism is still not clear, especially for the lack of a clear understanding of the mechanism of Han female estrogen derived endogenous skin aging. Moreover, the estrogen derived skin aging research is still mainly focused on single skin cells or skin (dermis, epidermis subunit or connective tissue) in the study area, little research on the whole the whole layer of the skin. In this study, the whole layer skin as the research object to explore genes related to estrogen induced skin aging and biological mechanism at the gene level.
Methods: We selected our hospital 13 elderly women patients with facial wrinkle surgery (age range, 46-55 years old, average 50.7 years old), according to the menstrual conditions were divided into 2 groups: premenopausal group and postmenopausal group. All patients were unified family history, history of diseases and other factors may exist in the waste. Patients were collected during surgery after the ear skin (light protection zone), full thickness skin after stored in liquid nitrogen, RNA extraction and further application of Illumina chip for whole genome scanning for genes. Appropriate statistical analysis of the results, and the gene function enrichment analysis and cluster analysis. The application of Q-RT-PCR on different gene further validation.
Results: we found 244 differentially expressed genes (P0.05). Among them, 131 genes up-regulated expression, downregulation of 113 differentially expressed genes. The G0 database for annotation of gene function and gene clustering analysis found that these differentially expressed genes were mainly involved in: metabolism, cell adhesion, metal ion binding, transcription, signal transduction, apoptosis, biosynthesis, ubiquitin cycle, immune response, inflammation, development and other biological processes. At the same time, we use the WebGestalt tools and KEGG database function enrichment analysis found that the difference of the 11 genes were enriched pathway genes, including lysine degradation, beta alanine metabolism, MAPK signaling pathway, insulin signal pathway, citrate cycle, RNA degradation pathway.
Conclusion: our study shows that endogenous estrogen and skin aging are closely related. This is a complex process involved by multiple genes and biological pathways. Some results from previous studies to validate each other, but there are still a lot of genes and biological pathways that have not previously done similar reports. We are considering to the skin as the research object and more objective response to biological network. Skin intrinsic aging results of our study will on estrogen induced skin aging related diseases and clinical study of estrogen replacement therapy. We only provide credible clues to genes and biological pathways has made a preliminary exploration and demonstration, still need more detailed and in-depth study to understand the specific mechanism of action.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R758.1

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