黑素小体降解对皮肤抵抗紫外线波长依赖光损伤和表皮钙梯度稳态影响研究

临床发现深肤色皮肤具有良好的抗紫外线（UVR）光损伤和屏障修复能力，被认为与黑素细胞（MCs）活性相关。位于表皮基底层的MCs加工生产充满黑素的被膜细胞器黑素小体，并借助树状突将这些色素化细胞器转移至毗邻的角质形成细胞（KCs），在KCs内黑素小体发生崩解或降解。黑素小体这一降解过程与KCs终末分化紧密偶联，推测影响着皮肤对UVR全波谱光保护和钙梯度依赖的屏障修复。本课题拟用蔗糖梯度离心法分离纯化黑素小体，用物理法使黑素小体崩解结合UVA诱导黑素分子氧化降解，观察被摄取完整或降解黑素小体的培养KCs或人皮肤替代物对UVA型/UVB型DNA损伤的抵抗能力。同时还观察体外荷载不同浓度钙离子的黑素小体对皮肤替代物中钙梯度形成的影响。本研究对认识转移后黑素小体降解影响皮肤光损伤光致癌的易感性，理解表皮钙梯度形成与维持机制以及指导临床构建色素化人工皮肤都有着重大意义。

The convincing clinical evidence is that there is enhanced photoprotection against ultraviolet radiation (UVR) and superior barrier repair functions in darkly pigmented skin, which are ascribed to melanocyte activity in the epidermis. In the basal layer, melanocytes synthesize melanin within membrane-bound organelles (melanosomes) and subsequently distribute those pigmented organelles to neighboring keratinocytes via their dendrites. Melanosomes are disintegrated and/or degraded in the recipient keratinocytes to release melanin pigments/dusts while ascending to the horny layer. Melanosome degradation is intimately coupled to the terminal differentiation of keratinocytes, which could contribute to full-spectrum UV protection and calcium-dependent skin barrier repair. In this study, we will isolate and purify the late-stage (III/IV) melanosomes from cultured melanocytes and MNT1 melanoma cells using an established sucrose-gradient centrifugation. Melanosomes are disintegrated first by a frozen/thawing method and then by manual grinding in a glass homogenizer, their melanin biopolymers enclosed in membrane-bound organelles are further degraded by UVA-inducd oxidation. We investigate the occurrences of UVA- or UVB-type DNA damages in cultured keratinocytes and reconstituted skin substitutes exposed to solar UVR under the broken or intact melanosome uptaking conditions. In addition, the modulations of calcium gradient in reconstituted skin substitutes containing the melanosomes with different calcium-loadings are visualized using a calcium-capture cytochemistry technique. Knowing the degradation of melanosomes in the epidermis would be the key to deciphering the skin susceptibilities to photodamage and photocacinogenesis, as well as unveiling the mechanisms of calcium gradient homeostasis. The proposed studies also provide clues that are of help in the reconstruction of tissue-engineered skin equivalents, effectively simulating human pigmented skin in vivo.