COL14A1基因在点状掌跖角化病发病中的作用及机制研究

Punctate palmoplantar keratoderma (PPPK) is a rare autosomal dominant skin disorder characterized by numerous hyperkeratotic papules, its pathogenesis is remained unknown, it is difficult to cure this disease, the physical and psychological health of patients was affected seriously. The applicant has performed exome sequencing analysis and identified the casual gene for PPPK- COL14A1,the achievement was selected for top ten science advance in chinese colleges and universities，2012 . Now, we plan to explore the role of this gene in the pathogenesis of PPPK based on previous findings. Firstly, we will construct the expression recombinant of COL14A1 gene and shRNA targeting against COL14A1, then the recombninants will be transfected into primary keratinocytes(KCs) to set up cell models with overexpression, mutation and low COL14A1 expression,then we will analyze the impact of COL14A1 on the proliferation and apoptosis of KCs, and further study the molecular mechanism that the COL14A1 acts on the KCs. In addtion, the mice of COL14A1 gene knock-out(COL14A1+/-) are develope to make the phenotypic expression of PPPK. The lesion of PPPK is biospied and biomarkers of proliferation and apoptosis are detected. Based on these studies, we try to elucidate the molecular mechanism of the of PPPK. This study may provide new ideas and clues for PPPK diagnosis and treatment.

点状掌跖角化病（PPPK）是一种单基因遗传性皮肤病，以皮肤过度角化增生为特征，发病机制尚不清楚，终身患病，严重影响患者身心健康。申请人前期利用全基因组外显子测序技术首次发现PPPK致病基因-COL14A1，成果入选2012年"中国高等学校十大科技进展"。本研究拟基于前期研究基础，通过构建野生型、 突变型COL14A1及shRNA表达载体，转染原代皮肤角质形成细胞（KCs），建立COL14A1表达调控模型，观察CO14LA1表达对细胞增殖、凋亡的影响，并研究其可能的分子机制；利用建立基因敲除小鼠，模拟疾病表型，借助皮损免疫病理，分析PPPK皮损中KCs的增殖、凋亡相关分子表达情况及可能的信号途径，揭示PPPK发病机制。为PPPK的诊断及治疗提供新思路。

本项目在前期利用全基因组外显子测序技术发现PPPK致病基因- COL14A1基础上，通过细胞培养，慢病毒载体构建，细胞转染，基因表达，通路分析等多种实验方法针对COL14A1基因与PPPK发病机制的关系进行了以下研究：1. 在原代培养的皮肤角质细胞（KCs）及HaCaT中发现存在胶原XIV（CXIV）蛋白表达提示COL14A1与PPPK存在显著关联性；2. 成功构建COL14A1基因野生型、突变型及shRNA的慢病毒表达载体，转染KCs及HaCaT，建立COL14A1表达调控模型，结果显示COL14A1过表达显著抑制KCs及HaCaT凋亡、增殖，COL14A1低表达显著促进KCs及HaCaT凋亡、增殖，与COL14A1低表达比较，COL14A1突变亦显著促进KCs及HaCaT凋亡、增殖，提示COL14A1基因突变导致皮肤角质细胞异常增殖，角化过度；3.将野生型和突变型的COL14A1基因分别构建入质粒载体pGEX-4T1，在原核表达系统中进行融合表达，从而亲和固化在谷胱甘肽亲和树脂上,作为与目的蛋白亲和的支撑物。将CD44构建入质粒载体pCDNA-3.1，体外翻译后通过层析柱或与此固相复合物混合，分离产物通过SDS-PAGE电泳分析，通过免疫共沉淀法证实CXIV与CD44存在相互作用，提示COL14A1基因的突变可能影响CXIV与CD44的相互作用，结果导致皮肤角质细胞异常增殖，角化过度，最终发展为PPPK； 4.提取野生型COL14A1过表达、突变型COL14A1过表达、COL14A1低表达及对照组的原代培养的皮肤角质形成细胞总蛋白，进一步研究发现COL14A1可通过线粒体、内质网应激凋亡途径及PI3K/Akt通路参与KCs凋亡的调节作用。