Mc1R通路在光疗促进毛囊神经嵴干细胞来源的黑素细胞系分化成熟及迁移中的作用及机制研究

Phototherapy is an important therapeutic option in the treatment of vitiligo, which initiated in a perifollicular repigmentation manner, supporting the follicular origin of the epidermal melanocytes. The hair follicle- neural crest stem cells（HF-NCSCs）were demonstrated the melanocyte-lineage differentiation potential. In our previous study, we found it could directly induce the mutation of the melanocyte lineage differentiated from HF-NCSCs with varied devices of phototherapy. Mechanistically, increased melanocyte maturation after phototherapy was resulted from increased expression of several key melanogenic factors, such as Mc1R, Sox10. Furthermore, we also found the 308-nm excimer laser or high intensity ultraviolet exhibited gerater efficiency on the maturation of melanocyte lineage differentiated from HF-NCSCs compared to the NB-UVB with a same dose, which was probably due to the stronger stimulatory action of Mc1r. Therefore we suppose Mc1R may display the pivotal role during the maturation of melanocytes derived from HF-NCSCs in the treatment of phototherapy. In this study, we will apply the molecular biology method for Mc1R knockout or high expression, use signaling pathway inhibitors/agonists, luciferase reporter gene to study the effects of Mc1R on the differentiation and migration for the melanocytes derived from HF-NCSCs in vitro with phototherapy. Furthermore we will investigate the underlying mechanisms about the detailed pathways mediated the signal transmission and the substrate mediated the transcription. Based on the foregoing, this study may provide important evidence for the molecular mechanism of vitiligo repigmentation induced by phototherapy and the development of new drugs for treatment.

光疗是临床治疗白癜风的重要手段，治疗后皮损的复色多呈现以毛囊为中心向四周扩展，提示与促进毛囊部干细胞向黑素细胞分化有关。而毛囊神经嵴干细胞（HF-NCSCs）被证实具有向黑素细胞等多种细胞系分化的潜能。我们的前期研究发现多种光疗仪器均可促进HF-NCSCs来源的黑素细胞系的分化成熟，并在此过程中上调黑素皮质素受体1（Mc1R）、Sox10等因子的表达，其中Mc1R在临床见效更快的308nm准分子激光或高能紫外线照射后表达上调较窄波紫外线更显著。据此我们推测Mc1R可能在光疗促进HF-NCSCs来源的黑素细胞系分化成熟中起关键作用。本研究拟应用Mc1R敲除或高表达，加入信号通路抑制或激动剂，以及荧光素酶报告基因等方法，探讨光疗作用下Mc1R对HF-NCSCs来源的黑素细胞系分化及迁移等影响，并明确其下游信号通路在胞内的传递和底物调控，从而为完善光疗治疗白癜风的机制及研发新型药物提供重要依据。

光疗是临床治疗白癜风的重要手段，治疗后皮损的复色多呈现以毛囊为中心向四周扩展，提示与促进毛囊部干细胞向黑素细胞分化有关。我们的前期研究发现多种光疗仪器均可促进毛囊部干细胞来源的黑素细胞系的分化成熟，并在此过程中上调黑素皮质素受体1（Mc1R）、Sox10等因子的表达，其中Mc1R在临床见效更快的308nm准分子激光或高能紫外线照射后表达上调较窄波紫外线（NB-UVB）更显著。据此我们推测Mc1R可能在光疗促进HF-NCSCs来源的黑素细胞系分化成熟中起关键作用。本项目研究首先建立了人来源的c-Kit(-)/CD57(-)皮肤毛囊干细胞的培养体系。通过NB-UVB光照证实光疗可有效促进人来源的c-Kit(-)/CD57(-)皮肤神经嵴干细胞向黑素细胞分化成熟；在微观形态学上分化的黑素样细胞表面的突触较未照光组有明显增加。而后我们应用Mc1R的shRNA病毒感染c-Kit(-)/CD57(-)人皮肤毛囊干细胞，观察Mc1R受抑制后的c-Kit(-)/CD57(-)人皮肤毛囊干细胞在NB-UVB照射下，分化细胞的酪氨酸酶（Tyro）表达量明显少于空载体组以及非目的基因对照组，证明Mc1R在光疗促进c-Kit(-)/CD57(-)人皮肤神经嵴干细胞来源的黑素细胞系分化成熟中起着重要作用。我们进一步在培养液中分别加入浓度合适的Mc1R下游信号通路中cAMP通路抑制剂H89、Erk通路抑制剂PD98089以及p38通路的抑制剂SB203580等进行研究，结果显示cAMP通路在光疗促进c-Kit(-)/CD57(-)人皮肤神经嵴干细胞来源的黑素细胞系分化成熟中起着重要作用。在分化细胞的迁移能力研究中，我们发现c-Kit(-)/CD57(-)人皮肤毛囊干细胞受NB-UVB低剂量（100mJ）照射后，分化细胞的迁移能力较未照光组增加，而高剂量（700mJ）照射后分化细胞的迁移能力较未照光组则下降。cAMP通路对分化细胞的迁移能力有轻度影响。Erk通路则表现为可显著影响分化而来的黑素样细胞的迁移能力。本项研究证实了Mc1R及其下游的cAMP通路在c-Kit(-) / CD57(-)人皮肤神经嵴干细胞向黑素细胞的分化成熟中有着重要作用；Erk通路则可显著影响分化的黑素样细胞的迁移能力。本研究结果为完善临床上光疗治疗白癜风的作用机制方面以及后续的研究提供重要依据和启示。