角蛋白1在溃疡性结肠炎发病机制中的作用

The morbidity of UC in China is increasing year by year and its pathogenesis is still unclear. Until now misdiagnosis and mistreatment of UC often happen in clinical practice due to lack of a gold standard for the diagnosis and effective treatment.Therefore,it is considered as a stubborn diseases by WHO. In recent years our team screens out differential expression of gene and protein by a sequence of previous study such as gene microarray, two-dimensional electrophoresis, mass spectrum analysis and so on in UC patients. Our preliminary research results revealed that the gene and protein of KRT1 were decreased expression in UC patients compared with the healthy comparers,which prompted that there was some correlation between KRT1 and pathogenesis of UC. However,there is no any study about the role of KRT1 in pathogenesis of UC so far.So we make a plan to find out this correlation.Firstly,to detect the expression of KRT1 on the intestinal mucosa of Crohn's disease, intestinal lymphoma, intestinal tuberculosis and infectious colitis to assess whether it is the biological activity maker of UC;then, to expand clinical cases for discussing the significance of the treatment and prognosis.Secondly, to reveal KRT1 occurrence and development of UC role at a cellular level by overexpression and RNA interference means . Finally ,to examine the effect of KRT1 to the gut barrier function and inflammation by UC experiment animal disease model using KRT1 overexpression genetic engineering mice. In brief,the research purpose is to illustrate whether KRT1 for UC markers, discover the roles of KRT1 in the pathogenesis of UC, and provide the novel theory and application basis for diagnosis and treatment of UC.

溃疡性结肠炎（ulcerative colitis,UC）的发病率逐年增高，发病机制未明，由于缺乏特异的诊断指标及根治措施常误诊误治，是世界性难题。本小组通过基因芯片、双向凝胶电泳及质谱分析等系列研究筛选出UC差异表达的基因和蛋白质，发现角蛋白1（keratin1,KRT1）在基因及蛋白水平均表达下调，提示KRT1与UC发病密切相关。目前国内外尚无KRT1与UC的研究报道，本项目通过；①检测KRT1在克罗恩病、肠淋巴瘤、肠结核、感染性肠炎中的表达，明确KRT1是否为UC诊断标志物；扩大UC病例探讨KRT1与UC严重程度的相关性；②质粒转染过表达和RNAi沉默干预Caco2细胞KRT1的表达，在细胞水平揭示KRT1在UC发生发展中的作用③建立KRT1过表达转基因小鼠UC模型，研究KRT1对结肠屏障功能及炎症的影响。旨在明确KRT1在UC发生发展中的分子机制，为诊治UC提供新的理论依据和方法。

我国溃疡性结肠炎（UC）发病率日益增高，目前本病的诊断主要依靠临床表现、肠镜和病理，尚缺乏判断疾病严重程度的特异性血清学指标，本小组前期的差异性基因和蛋白质研究发现角蛋白1（KRT1）为溃疡性结肠炎差异性基因，本项目进一步研究KRT1与UC的相关性及在发病机制中的作用。临床水平，通过免疫组化检测KRT1在克罗恩病、肠淋巴瘤、肠结核及感染性肠炎患者肠粘膜中的表达,发现KRT1为UC相对特异表达的蛋白质；采用免疫组化、PCR及Western blot检测KRT1在不同疾病严重程度UC患者肠粘膜中的转录和表达，证实KRT1表达水平与溃疡性结肠炎疾病严重程度呈负相关；动物水平,成功构建KRT1转基因小鼠，获得稳定的杂合子序列；KRT1转基因小鼠对DSS诱导的实验性结肠炎具有更高的敏感性，细胞骨架蛋白 KRT1与紧密连接蛋白claudin-2、occludin及ZO-1联合作用维持肠黏膜屏障功能，参与UC的发病过程。细胞水平，质粒转染上调KRT1表达后，Caco-2的紧密连接蛋白ZO-1表达升高，细胞极性增高，迁移率增高，细胞通透性降低，荧光素钠渗透率降低，IL-1β诱导的细胞凋亡率降低，但是KRT1过表达，不能抑制IL-1β诱导的促炎症因子TNF-α表达，提示KRT1在维持细胞膜屏障功能上起重要作用。这些研究证实KRT1在维持肠黏膜屏障中起着重要作用，为进一步研究其在UC诊断中的价值及治疗靶点提供理论依。