克罗恩病中干预TLE1逆转凋亡介导的肠道粘膜自噬紊乱的策略研究

Crohn’s disease is an multi-gene mediated autoimmune、chronic inflammatory disorder of the gastrointestinal tract. The pathogenesis of Crohn’s disease is complex with evidence to support both environmental and genetic factors. Currently the most in-depth research and analysis of Crohn’s disease pathogenesis involve in polymorphisms in NOD2、 autophagy variants and ER stress, which play the prominent role in the innate immune response towards intracellular bacteria. Our previous experiments through Whole-genome microarray found that the mRNA expression of TLE1 gene on the intestinal inflammatory mucosa of initial-onset Crohn’s disease patients was significantly lower than that of the normal people. After that, we preliminarily verified the reliability of microarray results in a small sample of Crohn’s disease cases. Based on existing numerous studies which have demonstrated the anti-apoptotic role of TLE1 gene in tumor diseases，We infer and will comprehensively verify that decreased expression of TLE1 gene in Crohn's disease cause decreased anti-apoptotic effects of intestinal epithelium through excessive activation of apoptosis-related proteins, resulting in aberrant intestinal mucosa autophagy-related gene expression and its dysfunction, thus upsetting the balance between the innate immunity, mucosal barrier homeostasis and intestinal microbiota in the intestinal mucosa of patients with Crohn’s disease. We will finally confirm this pathogenic hypothesis. Furthermore, through the intervention of TLE1 gene expression, it is possible for reversing the abnormal apoptosis mediated intestinal mucosa autophagy disorders. Thus patients with Crohn’s disease will possibly restore normal intestinal innate immune function. This study may expand new ideas for diagnosis and treatment of Crohn’s disease.

克罗恩病是一种多基因参与并协同作用下的复杂性、自身免疫性疾病，环境因素及遗传基因对其发病亦至关重要。目前克罗恩病发病机制研究最深入的即为NOD2多态性、自噬平衡紊乱及内质网应激三学说，而三者中的关键点又是自噬平衡紊乱学说。本课题组前期实验在表达谱芯片中发现初发型克罗恩病患者炎症部位TLE1基因表达量明显低于正常组人群，后在小样本病例中初步验证了芯片结果的可靠性。基于已有的大量研究证明该基因在肿瘤性疾病中抗凋亡方面的作用，我们推论并将全方位验证TLE1基因在克罗恩病患者的表达下降，令肠道粘膜抗凋亡作用下降而凋亡相关蛋白异常过度激活，致使肠道粘膜中自噬相关基因表达及功能异常，从而打破克罗恩病患者肠道粘膜先天免疫、粘膜屏障与肠道微生物群的稳态，最终确认这一致病假说。并通过干预TLE1基因表达，从而逆转凋亡异常介导的肠道粘膜自噬平衡紊乱，恢复克罗恩病患者正常肠道先天免疫功能，为该病的诊治拓宽新思路

克罗恩病是一种多基因参与并协同作用下的消化道慢性、非特异性、炎症性疾病，环境因素与遗传基因对其发病至关重要。其发病机制研究主要集中在NOD2基因多态性、自噬平衡紊乱及内质网应激三方面。自噬相关基因的表达与功能受损可导致肠黏膜自噬平衡发生紊乱。目前国内外对自噬相关基因的上位性调控基因的研究甚少，本研究旨在对活动期克罗恩病患者进行全基因组表达谱检测，筛选出可能与自噬相关基因存在相互作用的上游调控基因，以研究两者之间的生物学联系与内在作用机制，并深入探究干预上位基因后重塑肠黏膜自噬平衡的可行性。本研究纳入活动期克罗恩病患者及健康对照各4例，取患者病变结肠黏膜组织与健康对照正常结肠黏膜组织行Agilent 4*44K全基因组表达谱芯片检测，筛选出可能与自噬相关基因存在相互作用的差异基因，即TLE1，并进一步扩大样本量在40例活动期克罗恩病患者、43例活动期溃疡性结肠炎患者及37例健康对照的外周血单个核细胞及病变或非病变肠黏膜组织中以实时定量聚合酶链式反应或免疫组织化学染色进行验证。在HEK293、SW480及LoVo细胞中转染TLE1小干扰RNA或TLE1质粒，以蛋白质印迹法检测TLE1基因沉默或过表达后凋亡与自噬相关蛋白的变化，同时以FITC Annexin V/PI双染法与Hoechst染色法检测细胞凋亡情况，并采用免疫荧光技术在细胞与肠黏膜上皮组织中观察TLE1与自噬相关蛋白的定位与表达。以Ac-DEVD-CHO抑制TLE1干预后的凋亡水平，采用蛋白质印迹法检测自噬相关蛋白的相应变化以证实凋亡途径在TLE1与自噬间的介导作用。研究结果表明TLE1基因表达降低与活动期克罗恩病相关，且具有克罗恩病特异性。该基因的低表达可促进细胞凋亡并通过上调Cleaved caspase-3与Cleaved caspase-9的表达介导ATG16L1降解与自噬抑制。抑制TLE1的低表达或可能逆转过度凋亡的发生从而恢复自噬水平。TLE1可能通过凋亡途径对ATG16L1进行上位性调控，该基因或可能成为克罗恩病新型生物标志物与发病机制研究新靶点。