调控YAP基因对银屑病角质形成细胞增殖的影响

The pathogenesis of psoriasis is not fully elucidated, abnormal expression of various signal pathways was involved in the development of psoriatic lesions. Hippo-YAP signaling pathway play a key role in the regulation of cell proliferation, differentiation and organ size. Our preliminary study has shown increased YAP expression in psoriatic lesions. Therefore we hypothesize that the Hippo-YAP signaling pathway may be involved in the pathogenesis of psoriasis. In this application, we propose to examine the role of YAP, a key molecule of Hippo-YAP signaling, in the regulation of keratinocyte proliferation and its significance in the psoriasis using immunologic and molecular biologic methods. We will use RNA interference, real-time RT-PCR, Western blot technology and immunstaining to evaluate the effect of YAP on the cytokine profile and its association with WNT signaling in IL-22 induced primary cultured keratinocyte, and its expression in the lesions of psoriasis. We will also explore if YAP contribute to the imiquimod-induced psoriasis like lesions in animal model. Successful completion of current proposal will further advance our understanding of the pathogenesis of psoriasis, and also help to further deepen the understanding of Hippo-YAP signaling pathway.

银屑病的发病机制目前尚未完全阐明，信号转导通路异常在银屑病的皮损形成中发挥关键作用并与银屑病的免疫机制息息相关，Hippo-YAP信号通路是近年来倍受关注的一个调控细胞增殖凋亡和组织器官大小的关键信号途径，我们前期研究也证实YAP蛋白在银屑病皮损中处于高表达状态，由此提出Hippo-YAP信号通路可能在银屑病发病机制中起重要作用。本项目拟运用RNA干扰、real-time RT-PCR、western blot等技术手段，从组织标本原位分析、细胞培养到动物模型实验，探讨YAP在银屑病皮损中的激活状态及调控YAP对IL-22诱导的原代角质形成细胞生长、增殖、凋亡及分泌炎症因子的影响，并初步了解YAP与银屑病相关信号通路Wnt交互作用。本研究将为系统阐明Hippo-YAP信号通路在银屑病发病、治疗中的作用提供研究基础，也有助于进一步加深对Hippo-YAP信号的认识。

银屑病的发病机制目前尚未完全阐明，信号转导通路异常在银屑病的皮损形成中发挥关键作用并与银屑病的免疫机制息息相关，Hippo-YAP信号通路是近年来倍受关注的一个调控细胞增殖凋亡和组织器官大小的关键信号途径，我们前期研究也证实YAP蛋白在银屑病皮损中处于高表达状态，由此提出Hippo-YAP信号通路可能在银屑病发病和治疗中起重要作用。本项目拟运用RNA干扰、ELISA、western blot等技术手段，从组织标本原位分析、细胞培养到后期动物模型，探讨YAP在银屑病皮损中的激活状态及调控YAP对角质形成细胞生长、增殖、凋亡及分泌炎症因子的影响，并初步了解YAP与银屑病相关信号通路Wnt交互作用。.主要结果和关键数据：（1）免疫组化结果显示YAP、磷酸化YAP（P-YAP）在银屑病皮损中表达水平增高（2）Hacat细胞培养，构建慢病毒，干扰细胞中YAP1基因表达，western blot实验确认干扰效果。（3）基因芯片检测慢病毒转染前后对细胞分泌细胞因子的影响及各个信号通路的影响，从基因水平筛选数据。（4）慢病毒感染前后对细胞周期和细胞凋亡的影响，实验结果：相比对照组shYAP1组处于S期的细胞经T-Test分析P>0.05，处于G1期的细胞经T-Test分析P<0.05，处于G2/M期的细胞经T-Test分析P<0.05。相比对照组，shYAP1组凋亡数经T-Test分析P<0.05。（5）用Western blot检测调控前后细胞中FAS、caspase3及caspase8表达水平的变化和细胞周期蛋白CyclinD1、CyclinA及CyclinE的变化。（6）ELISA方法检测YAP基因不同激活状态下，角质形成细胞分泌相关细胞炎症因子IL-17、IL-6、TNF-a及VEGF蛋白的表达。（7）构建咪喹莫特小鼠模型成功，观察到YAP siRNA 体内注射对咪喹莫特诱导银屑病样皮损有抑制效应。.本研究将为系统阐明Hippo-YAP信号通路在银屑病发病、治疗中的作用提供研究基础，也有助于进一步加深对Hippo-YAP信号的认识。