Gal-9在维持肠上皮屏障功能中的作用机制研究

The incidence of Inflammatory Bowel Disease (IBD) in China has been gradually increasing, but the exact etiological factor and mechanism remains unclear, our knowledge about how epithelial barrier function is compromised in IBD is still limited. Research demonstrate that the intestinal epithelial barrier defects and dysfunction is the pathological basis. Abnormal immune response is closely associated with IBD. The skewed Th1/Th17 cellular immune responses play an important role in the development of IBD. It has been shown that Lectin 9 (Gal-9) can inhibit the release of cytokines from Th1/Th17 cell. However, it is not known whether Gal-9 is the important factor to induce skewed Th1/Th17 cellular responses and involved in the development of intestinal inflammation,this question need to further investigate. Our preliminary results revealed that Gal-9 plays a critical role in sustaining allergic status in mouse intestine; and the expression of Gal-9 is significantly weaker in IBD intestinal mucosa than in non-IBD intestinal tissue. We hypothesis that IEC-derived Gal-9 is suppressed and it could exacerbate the intestinal inflammation. A panel of in vivo experiments(with Transwell coculture System) and in vitro experiments(with TNBS mouse colonic inflammation model) will be performed to investigate the effect of Gal-9 on the restoration of intestinal epithelium by RNAi to knock out target gene, Gal9-expression lentivial vector to transduce IEC and Ussing chamber technique to examine the barrier function in colon epithelial layer of mice. Also,other experimental methods will be used in this research. The results from this project will elucidate the role of Gal-9 in maintaining the intestinal epithelium barrier function and provide fundamental information for designing a new gene therapy target for treatment of IBD.

我国炎症性肠病(IBD)发病率逐年增高，其确切病因和发病机制仍不清楚。研究表明，肠黏膜屏障受损及功能失调是其病理基础，异常免疫反应与其发病密切相关。Th1/Th17细胞免疫失衡与IBD发生有关。半乳糖凝集素9(Gal-9)能够阻止Th1/Th17细胞因子的释放。Gal-9是否能通过抑制Th1/Th17细胞应答而抑制肠道炎症，从而维持肠上皮屏障功能值得深入研究。我们前期研究发现，小鼠肠上皮细胞(IEC)表达Gal-9并在食物过敏肠黏膜损伤修复中起重要作用；IBD患者IEC表达的Gal-9明显降低。我们假设：IEC表达的Gal-9可能与肠道炎症发生关系密切。本课题将通过Transwell细胞实验和动物实验，利用基因敲出，慢病毒载体转染，Ussing Chamber等技术来探讨Gal-9在肠黏膜损伤及修复中的作用。以期阐明Gal-9在维持肠上皮屏障功能中的机制并为IBD的基因治疗提供理论依据。

本课题为1年期面上项目。由于时间和经费的限制，我们仅对Gal-9肠上皮细胞的作用做了初步探讨。采用Transwell细胞共同培养系统，利用ELISA、免疫细胞化学、RNAi、FCM、慢病毒转染等技术方法研究微生物产物、促炎细胞因子等可能影响T84表达Gal-9的因素。后续研究中我们将还要按课题设计内容通过栋实验探讨Th1/Th17相关细胞因子对Gal-9表达及黏膜屏障的影响；特别是慢病毒载体转染结肠上皮细胞促使其过量表达Gal-9以修复肠道黏膜屏障、恢复肠黏膜屏障功能的研究。.我们的初步研究结果显示： IBD患者血清及肠粘膜Gal-9水平均降低并与病情轻重均呈正相关性；Th1特异性细胞因子IFN-γ，TNF-α以及Th17特异性细胞因子IL17，IL23水平变化及动物实验结果初步显示也与IBD患者相一致；体外实验显示：免疫细胞化学和RT-PCR均证明T84细胞表达Gal-9；细菌鞭毛蛋白（Cbir1）能够抑制T84细胞Gal-9的表达；LPS及PGN的干预，慢病毒载体mGal9Lv转染小鼠肠上皮细胞实验正在进行中。