Elucidating melatonin-regulated mechanisms of melanogenesis in human full-thickness skin and human melanocytes exposed to ultraviolet radiation

与皮肤色素沉着相关的临床疾病包括色素沉着或色素沉着过多，因此强烈需要活性和安全的色素调节剂。基于迄今为止所描述的褪黑素的高生物活性，它可能是一种有效控制黑色素形成机制的有效物质。褪黑素（n -乙酰-5-甲氧基色胺）是脊椎动物松果体的分泌产物，调节多种功能，包括清除自由基，DNA修复或抗凋亡活性。它还涉及皮肤功能，如表皮增殖/分化，头发生长和黑色素瘤/鳞状细胞癌的控制。本研究拟探讨褪黑素对黑色素形成调控机制的潜在影响。到目前为止，申请人在褪黑素领域获得的广泛经验使他能够进行本提案中所包含的所有实验。计划中的研究将在人类离体全层皮肤（第一部分）中进行，第二部分使用纯培养的正常人类黑素细胞（NHM），与与正常人类角质形成细胞（NHEK）共培养进行比较，后者将反映体内皮肤生理。此外，这种方法可以描述角化细胞-黑素细胞的相互作用，以及角化细胞依赖性调节黑素形成的关键作用。在皮肤中，褪黑激素的作用和作用将从uvr介导的黑色素形成方面进行评估，包括褪黑激素对酪氨酸酶（TYR）的影响，TYR是黑色素形成的关键酶之一，控制微白相关转录因子（MITF）的表达，褪黑激素对黑色素合成和cAMP-PKA-MITF轴的影响。此外，我们将对前面提到的NHM、NHEK细胞系进行一组分析，这部分与第一个细胞系的数据一起，对褪黑素调节的黑色素形成的分子作用方式有一个全面的了解。在这两种实验模型中，为了研究褪黑激素是否在这些基因发生改变的情况下维持这一过程，将对黑素生成依赖基因进行沉默。结果无疑将对褪黑素在UVR条件下皮肤黑色素形成中的作用提供全新的认识，并为色素疾病的治疗提供新的原则。

Clinical disorders related to skin pigmentation include hypo- or hyperpigmentation, therefore active and safe pigment modulatory agents are strongly desired. Based on the so far described high biological activity of melatonin, it can be a potent substance that effectively controls mechanisms of melanogenesis. Melatonin (N-acetyl-5-methoxytryptamine) is a secretory product of the vertebrate pineal gland modulating various functions including scavenging free radicals, DNA repair or anti-apoptotic activity. It is also implicated in skin functions such as epidermal proliferation/differentiation, hair growth and melanoma/squamous cell carcinoma control. In this research application, it is proposed to evaluate the potential impact of melatonin on regulatory mechanisms of melanogenesis. So far wide-range experience gained by the applicant in the field of melatonin allows him to conduct all experiments enclosed in this proposal. The planed investigations will be carried out in human ex vivo full-thickness skin (PART I) with additional PART II using normal human melanocytes (NHM) in pure culture in comparison to co-culture with normal human keratinocytes (NHEK) that will reflect skin physiology in vivo. Additionally, such approach may describe the keratinocyte-melanocyte interaction as well as the key role of keratinocyte-dependent regulation of melanogenesis. In skin, the role and action of melatonin will be evaluated with respect to UVR-mediated melanogenesis including melatonins impact on tyrosinase (TYR), one of the key enzymes in melanogenesis, controlling the expression of microphtalmia-associated transcription factor (MITF) as well as the effect of melatonin on melanin synthesis and cAMP-PKA-MITF axis. Furthermore, a set of analysis will be performed with the earlier mentioned cell lines, i.e. NHM, NHEK, and this part together with data obtained from the first one is supposed to provide a comprehensive knowledge over the molecular way of action of melatonin-regulated melanogenesis. In both, experimental models, silencing of melanogenesis-dependent genes will be performed in order to investigate whether melatonin may maintain this process in case of alterations within these genes. Resulting findings undoubtedly will put in a brand new insight into the role of melatonin in cutaneous melanogenesis under UVR conditions and provide new principles for treatment of pigmentary disorders.