POFUT1基因突变导致色素异常分子机制研究

Pigmentation disorders belong to a common type of skin disease. Studies on the genetic mechanisms of pigment metabolism bring benefit to our understanding of the pathogenesis at the molecular level of this group of skin diseases. Using a genome-wide linkage analysis and exome sequencing, our previous study has identified that protein O-fucosyltransferase 1 (POFUT 1) gene is the pathogenic gene for generalized Dowling-Degos disease. Moreover, protein O-fucosyltransferase 1 plays an important role in the conserved Notch signaling pathway. We have also constructed a zebrafish model for generalized Dowling-Degos disease by using morpholino antisense pofut1 gene silencing technique. The current project plans to construct recombinants of POFUT1 gene and shRNA targeting against POFUT1, which will be transfected into primary keratinocytes and melanocytes to set up cell models with over or lower expression of POFUT1, respectively. A vitro model of keratinocyte and melanocyte co-culture system is then established for studying the role of POFUT1 gene and its Notch signaling pathway played on growth and differentiation of melanocytes and keratinocytes, as well as synthesis and transportation of melanosomes. Meanwhile, we will use the constructed zebrafish model to investigate the impact of down-regulation of pofut1 expression on intercellular connection of keratinocytes, as well as the amount, distribution, differentiation,and migration of melanocytes.The fundamental purpose of the research is to reveal the possible molecular mechanism of pigmentation anomalies and provide new ideas for prevention and treatment of the pigmentation disorders.

色素异常性疾病是一类发病率较高的皮肤病，针对色素异常代谢的分子机制研究将有助于我们对此类疾病发病机制的理解。在前期研究中，我们利用全基因组关联分析和外显子组测序技术，发现了泛发性屈侧网状色素异常的致病基因为蛋白O岩藻糖基转移酶1基因（POFUT1），它是高度保守的Notch信号通路的重要组成部分，同时利用反义吗啡啉基因沉默技术成功建立了POFUT1基因沉默的斑马鱼疾病模型。本项目拟构建野生型POFUT1及shRNA表达载体，转染黑素细胞及原代角质形成细胞，并将两者进行共培养，研究该基因及其所在Notch信号通路对黑素细胞和角质形成细胞生长和分化以及黑素合成和转运的影响，同时利用已经建立的斑马鱼疾病模型，进一步研究下调该基因表达对斑马鱼皮肤中角质形成细胞的连接以及黑素细胞的数量、分布、分化和迁移的影响。通过以上研究旨在揭示皮肤色素异常的可能发病机制,为色素异常性疾病的防治提供新的思路。

本课题前期研究发现泛发性屈侧网状色素异常的致病基因为POFUT1基因，同时利用反义吗啡啉基因沉默技术成功建立了POFUT1基因沉默的斑马鱼疾病模型。本研究旨在研究POFUT1基因突变导致色素异常的分子机制，研究发现在电镜下观察到患者皮损中黑素细胞数量偶见，存在少量胞突，胞内无色素颗粒，表明POFUT1基因功能缺陷严重影响人体黑素细胞内黑素的合成或转运障碍。患者皮损组织qRT-PCR检测和免疫组化均显示POFUT1、NOTCH3、MITF、TYR、KRT5表达不同程度降低，HMB45蛋白在皮突延长处呈阳性。利用RNA干扰敲除POFUT1 慢病毒感染原代黑素细胞与原代黑素细胞比较，发现RNA干扰敲除POFUT1的黑素细胞在Notch信号通路上，Notch1受体、Notch3受体表达显著性降低，黑素运输相关基因MLPH 表达显著性降低。POFUT1基因突变导致泛发型屈侧网状色素异常患者皮肤黑素小体成熟障碍，与调控PMEL17基因相关，提示POFUT1基因可能通过Notch3受体激活信号通路下游靶基因影响皮肤色素的合成和转运。利用透射电镜和扫描电镜观察斑马鱼疾病模型角质形成细胞的形态和数量无明显异常，黑素细胞数量无明显变化，但其中黑素小体的分布存在明显的不均匀，提示POFUT1基因功能的缺陷，影响黑素小体的合成和转运斑。斑马鱼的 RNA rescue 试验，证实人源性的 POFUT1 基因可以挽救斑马鱼疾病模型，进一步验证 pofut1 基因对于黑素合成的重要性。同时在该研究经费的支持下，我们对近400例遗传性色素异常性皮肤病致病原因进行研究。本研究已发表标注相关论文14篇，1篇有待发表。

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