氧化应激下角质形成细胞来源外泌体通过释放miRNAs诱导黑素细胞凋亡和功能障碍参与白癜风发病的作用和机制研究

Vitiligo is a skin disease characterized by the desctruction of epidermal melanocytes. Oxidative stress is closely related to the development of vitiligo. As the main constituent cells in the epidermis, keratinocytes regulates the viability and the function of melanocytes, but the underlying mechnism is still not clear. Our previous studies have shown that keratinocytes could secret exosomes under oxidative stress, which induces the apoptosis of melanocytes. Moreover, miR-16, a miRNA that can inhibit the expressions of multiple target genes involved in the survival and the function of melanocyte, is significantly overexpressed in the exosomes derived from keratinocytes under oxidative stres, indicating that keratinocytes-derived exosomes play a key role in the death and the dysfunction of melanocytes under oxidative stress in vitiligo. Based on these findings, we hypothesize that in vitiligo, keratinocytes secret exosomes under oxidative stress, which can enter into adjacent melanocytes in a paracrine way and release functional miRNAs including miR-16, subsequently facilitating the death and the dysfunction of melanocytes via supressing target genes and ultimately leading to the disappearance of epidermal melanocytes and the formation of skin depigmentation. The project plans to clarify the effects of exosomes derived from keratinocytes on oxidative stress-induced melanocyte destruction as well as the involved mechanisms at molecular, cellular, animal and clinical levels, which may provide new theoretical basis for the treatment of vitiligo.

白癜风是一种以表皮黑素细胞（MC）破坏为主要特征的皮肤病，氧化应激在其发病中发挥重要作用。作为表皮的主要组成细胞，角质形成细胞（KC）在调控MC的活性和功能方面发挥重要作用，但机制不清。我们的前期研究发现，氧化应激下KC释放外泌体，作用于MC后诱导其凋亡；进一步分析发现miR-16在氧化应激下KC来源外泌体中表达水平显著升高，可抑制多个黑素细胞生存和功能相关靶基因的表达，提示KC释放外泌体是白癜风氧化应激下黑素细胞出现死亡和功能障碍的关键事件。基于以上我们提出研究假说：白癜风氧化应激下KC产生外泌体，以旁分泌形式进入邻近黑素细胞，释放miR-16等活性miRNAs分子，通过抑制靶基因表达诱导MC死亡和功能障碍，最终导致MC的破坏消失和皮肤白斑的形成。本研究拟在分子、细胞、动物和人群水平系统阐释氧化应激诱导KC来源外泌体致黑素细胞破坏过程中的关键作用和机制，为白癜风的治疗提供新的理论依据。

白癜风是一种以表皮黑素细胞破坏为特征的皮肤病。氧化应激在其发病中具有重要作用。作为表皮的主要组成细胞，角质形成细胞通过诱导黑素细胞死亡或招募抗原特异性CD8+T细胞杀伤黑素细胞介导氧化应激对黑素细胞的破坏。角质形成细胞分泌大量具有生物学功能的外泌体，但这些外泌体对黑素细胞存活及对CD8 +T细胞活性的影响不清楚。本研究提取正常培养及H2O2刺激下角质形成细胞上清外泌体并用不同方法进行鉴定；在体外和白癜风小鼠模型中研究它们在黑素细胞、CD8+T细胞及小鼠白癜风发生发展中的功能；用小RNA-seq筛选并鉴定Exo.T富含的miRNAs并在体外研究这些miRNAs在黑素细胞和CD8+T细胞中的功能及机制；最后，在临床样本中研究Exo.T富含的miRNAs与白癜风进展的关系。结果显示，我们从角质形成细胞上清提取的外泌体为直径60至200nm的双层膜囊泡，表达外泌体标记蛋白CD9、CD63、TSG101、Alix和HSP70，证明所提为外泌体。氧化应激促进角质形成细胞外泌体的分泌。Exo.T具有抑制黑素细胞存活、促进 CD8 + T细胞增殖、活化及IFN-γ分泌、促进小鼠白癜风进展的功能。Exo.T具有特异的miRNAs表达谱，富含miR-1246、miR-31-3p、miR-374b-5p及miR-139b-3p。miR-31-3p可能通过抑制MITF信号轴破坏黑素细胞抑制黑素生成，而其在CD8+T细胞活化中的促进作用可能通过多条途径。miR-31-3p在进展期白癜风患者血清中高表达，它可能促进白癜风的发生发展。综上，我们的研究表明氧化应激下角质形成细胞所分泌的富含miR-31-3p等 miRNAs的外泌体通过破坏黑素细胞并诱导CD8+T细胞活化促进白癜风的进展。本研究阐释了氧化应激诱导角质形成细胞来源外泌体导致黑素细胞破坏过程中的关键作用和机制，可为白癜风的治疗提供新的理论依据。