PIK3C3突变导致上皮完整性破坏和炎症反应的机理研究

The epithelial barrier function is one of the important features of intestinal tissue. However, damages to epithelial integrity may lead to Inflammatory Bowel Disease (IBD). In the zebrafish model and the in vitro 3D cell model, we initially demonstrated that by pik3c3 knockdown intestinal epithelial tissue integrity was disrupted and ultimately lead to inflammatory responses. To address the causes of mutant inflammatory phenotypes, we hypothesized that due to the deficiency of pik3c3, cell junction-associated proteins could not maintain homeostasis through normal vesicle trafficking and epithelial cell junctions would be destroyed, leading to abnormal epithelial barriers and inflammatory responses. Therefore, this project intends to study the pathogenic mechanisms from the following aspects: (1) the relationships between cell junction-associated membrane protein recycling transport and epithelial integrity maintenance; (2) the relationships between epithelial integrity damage and inflammatory response; (3) evaluation of IBD drugs using zebrafish mutants. This study will not only enrich the function of PIK3C3 in the field of organ development and disease, but also provide a model for epithelial autonomous injury for IBD drug evaluation.

上皮屏障作用是肠道组织的重要特征之一,而上皮完整性的破坏可能会导致炎症性肠病（Inflammatory Bowel Disease, IBD）。在斑马鱼模型和体外3D细胞模型中，我们初步证明pik3c3敲除会破坏肠道上皮组织完整性，并且最终导致突变体发生炎症反应。针对突变体炎症表型产生的原因，我们提出假设：由于pik3c3缺失，细胞连接蛋白不能通过正常的囊泡运输保持稳态，最终破坏了上皮细胞连接，导致上皮屏障作用异常和炎症反应。因此，本项目拟从以下方面研究致病机理：(1)细胞连接相关的膜蛋白循环运输与上皮完整性维持的关系；（2）上皮完整性损伤与炎症反应的关系；(3)利用斑马鱼突变体进行IBD药物的评估。本研究不仅将丰富PIK3C3在器官发育和疾病领域的功能，还为IBD药物评估提供了上皮自主性损伤的模型。

炎症性肠病（Inflammatory Bowel Disease，IBD）属于自身免疫性疾病，其致病机理尚未清楚并且不能完全治愈。根据前期的研究结果，斑马鱼pik3c3突变体表现出了肠道上皮完整性破坏、大量中性粒细胞聚集等IBD的主要特征，适合研究IBD的发病机理和治疗方法。按照申请书的研究内容和研究计划，我们得到以下结果：（1）细胞连接蛋白等膜蛋白的动态平衡受到胞内体-多泡体-循环胞内体/溶酶体调控，PIK3C3突变会导致E-Cadherin等细胞连接蛋白聚集在多泡体中；（2）斑马鱼pik3c3突变体肠道组织蛋白质、脂类和糖类等营养物质吸收和代谢过程受到抑制；（3）斑马鱼pik3c3突变体肠道组织TLR（Toll样受体）等天然免疫被显著激活；（4）JAK/STAT通路抑制剂Tofacitinib、广谱免疫抑制剂Rapamycin等多种IBD药物可以抑制炎症反应的激活来缓解PIK3C3突变体部分表型。在斑马鱼pik3c3突变体中，即使炎症反应被抑制到较低水平，肠道粘膜上皮极性仍然受破坏。综上所述，本项目不仅解释了PIK3C3突变、上皮完整性缺失以及炎症反应的关系，还为IBD的发病机理研究以及药物研发提供了新的思路。