IL-23/IL-23R/IL-17新型免疫通路与黏附侵袭性大肠杆菌LF82在克罗恩病发病机制中的研究

Crohn's disease (CD) is a chronic relapsing and remitting inflammatory condition which affects any part of the gastrointestinal tract, mainly affecting the gastrointestinal tract with extraintestinal manifestations and associated immune disorders. More and more patients in China during the last twenty years, and badly affect the quality of people's lives. Several experiments have led to the theory that the pathogenesis of CD is the result of an aberrant immune response to normal commensal bacteria in genetically susceptible individuals. Genome wide association studies (GWAS) identified susceptibility loci that triggered by environmental factors, and result in a disturbed innate and adaptive immune response towards a diminished diversity of commensal microbiota. One of the strongest gene associations in CD observed in GWAS studies is the gene encoding interleukin-23 receptor (IL-23R), the IL-23/IL-23R/IL-17 pathway has recently been identified to play a critical role in a number of chronic inflammatory diseases including CD. The identification in CD patients of associations in IL23R has highlighted the importance of proper IL-23/IL-23R /IL-17 pathway regulation in intestinal immune homeostasis. In addition, several lines of investigation have associated microorganisms with stimulating the inflammation associated with Crohn's disease. Adherent invasive Escherichia coli (AIEC) have been demonstrated to colonize the intestinal mucosa of patients suffering with Crohn's disease.However, till now, nothing has been reported about the relationship between susceptibility gene of CD and AIEC. We aim to study the relationship between the single nucleotide polymorphism in IL-23R and effect of AIEC strain LF82, and then explore the role of IL-23/IL-23R/IL-17 pathway in the pathogenesis of CD exacerbated by LF82. Combine together the genetic factor, immune factor and microbiotic factor, and to provide a new perspective in the study of CD pathogenesis.

克罗恩病（CD）是一种可累及全消化道的慢性复发性炎症性疾病，近20年来我国发病越来越多，严重影响患者生命健康。研究认为CD是遗传易感个体对肠道微生物的过度免疫反应。全基因组关联分析（GWAS）已鉴定出多个CD易感位点，其中IL-23R基因与CD之间联系明显。近年来IL-23/IL-23R/IL-17新型免疫通路的阐明为CD的研究提供了新思路。此外，研究还认识到肠道微生态对CD的重要性，其中侵袭黏附性大肠杆菌（AIEC）可定植在CD患者肠粘膜，可能是CD发病的始动和持续因素。到目前，CD的易感基因与AIEC之间是否存在协同作用尚无报道，本课题拟通过将CD可能的易感基因IL-23R与AIEC菌株LF82相联系，探讨IL-23/IL-23R/IL-17通路与AIEC菌株LF82在CD发病中的作用和机制。将CD发病的遗传因素、免疫因素和肠道微生态因素相结合，为研究CD的发病机制提供一个新视角。

以往的文献报道对IL-17在小鼠结肠炎症中究竟是促进炎症还是抑制炎症存在着争论，但这些文献都忽略了肠道微生态的影响。我们研究在有特定致病菌定植的肠道环境中，IL-17所扮演的角色。.主要研究结果：1.通过HE和病理学评分发现定植的AIEC可导致野生型小鼠结肠上皮细胞轻微紊乱和炎性细胞浸润明显，通过PCR和免疫组化发定现其结肠组织中IL-17的分泌量升高。相比定植AIEC的野生型小鼠，IL-17敲除鼠定植AIEC后结肠上皮细胞更紊乱，炎性细胞浸润也更加明显。通过电镜显示定植AIEC以后，野生型小鼠肠上皮细胞微绒毛排列有序，而IL-17敲除鼠结肠上皮细胞微绒毛大量脱落，细菌以胞吞的方式侵袭到结肠上皮细胞。2.通过急性活动相关性指标评价发现AIEC并没有加重DSS诱导野生型小鼠结肠炎症。在DSS诱导野生型小鼠结肠炎症中，PCR和免疫组化发现AIEC使结肠中IL-17含量升高。接着通过急性活动相关性和组织学评分发现，相比定植AIEC的野生型小鼠，AIEC对DSS诱导IL-17敲除小鼠的结肠炎症程度加重更明显。3.通过检测结肠组织炎症因子发现，在普通肠道AIEC的定植使IL-17敲除小鼠结肠中IL-22的分泌降低。在小鼠实验性结肠炎中，AIEC可进一步降低DSS诱导IL-17敲除小鼠结肠中IL-22的分泌释放。说明IL-17能够促进IL-22分泌。.结论：我们的研究证实AIEC可刺激野生型小鼠结肠组织中IL-17分泌的升高；而在IL-17缺乏时，可破坏小鼠肠上皮屏障完整性。在DSS诱导实验性野生型小鼠结肠炎中，AIEC也可刺激小鼠结肠IL-17分泌增加；而在IL-17敲除小鼠中，AIEC也可加重DSS诱导的小鼠结肠炎症，表明在IL-17缺乏时，AIEC可加重炎症；在定植AIEC后，与野生型小鼠小鼠相比，IL-17敲除小鼠DSS诱导炎症程度加重，表明在AIEC定植后，IL-17缺乏时可加重炎症。这就从正反两面表明，IL-17在定植了AIEC的DSS诱导的炎症中有保护作用。进一步研究其中的机制发现IL-17能够促进炎症保护性细胞因子IL-22分泌，共同保护结肠肠上皮屏障。