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

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.

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。

【摘要】 项目背景 黑素小体（melanosome）自黑素细胞（MC）向毗邻角质形成细胞（KC）转移被认为是重要的皮肤光保护机制之一。随着终末分化KC自表皮基底层向角质层迁移，被转移至KC内的黑素小体也同步发生降解。然而，黑素小体降解前后光保护、抗氧化等生物学活性是否改变至今仍缺乏认识。研究内容 1）从原代培养人MC或MNT1人黑素瘤细胞株分离纯化黑素小体，用物理法（反复冷冻-复苏联合组织研磨）破碎黑素小体，比较完整与破碎黑素小体对UVA或UVB诱导KC光损伤的保护作用与羟自由基清除能力；2）通过提高培养基钙离子浓度诱导KC分化，测定组织蛋白水解酶V（CTSV）的蛋白表达水平与酶活性，比较被吞噬黑素小体在（高钙）分化KC与（低钙）未分化KC内黑素小体的降解速率；3）用qPCR技术测定脂溢性角化病皮损中CTSV表达水平以及测定表皮裂解物对体外孵育纯化黑素小体超微结构的变化；4）用四氧化钌电子特染结合透射电镜技术比较观察白癜风无色素皮损与正常皮肤角质层板层脂膜的超微结构，用钙离子捕捉技术结合透射电镜技术观察二者表皮钙沉淀颗粒的数量和分布；5）分析了氢醌及其糖苷衍生物（脱氧熊果苷）对黑素小体超微结构的影响以及3种木质素降解酶对黑素的脱色作用。数据与结果 1）与破碎黑素小体比较，完整黑素小体显示较强的光保护与抗氧化活性；2）高钙诱导分化的KC高表达CTSV，后者参与KC内被转移黑素小体的降解。而UVB照射KC可以下调CTSV的表达，提高完整黑素小体与破碎黑素小体的比例，并增强了皮肤UVA/UVB抵抗能力；3）脂溢性角化病皮损存在CTSV表达降低与黑素小体降解障碍，这一现象可能与该病的病理发生相关。白癜风脱色素皮损（一种体内黑素小体缺失或降解模型）存在板层脂膜超微结构和表皮钙离子分布紊乱；4）脱氧熊果苷显示出较强的皮肤增白活性，且缺乏对黑素小体的直接毒性。科学意义 对黑素小体降解及其调控机制的认识，有助阐释色素性皮肤病的发病机制以及研发不消弱皮肤光保护能力的新型皮肤色素调节剂。