紧密连接蛋白异常磷酸化对结肠粘膜屏障功能的影响及其机制研究

Ulcerative colitis (UC) is a chronic inflammation in the mucosa,which seriously affecting the quality of life .Intestinal barrier dysfunction takes an important part in the development of UC. Tight iunction is the basis of intestinal mucosal barrier. Preliminary experiments found differential expression of tight junction proteins in UC mice, paralleled with intestinal mucosal barrier disfunction. As reported in previous stusies, abnormal phosphorylation of tight junction proteins leading to the changes of polarity and allosterism, thus affect the epithelial barrier function. Whether the tight junction protein was phosphorylated in UC intestinal mucosa, remains largely undefined. We assume that the abnormal phosphorylation of intestinal tight junction protein affecting the permeability of intestinal epithelial cells, delaying the wounding-healing process, resulting in intestinal mucosal barrier dysfunction. In the present study, we firstly observe the phosphorylation of tight junction protein in the UC mucosa. By the means of ultraphosphorylation and dephosphorylation, we also observe the changes of intestinal epithelial cell proliferation/apoptosis, injury/repair and paracellular permeability, further explained the pathogenesis of UC, and the mechanism for abnormal phosphorylation of tight junction proteins. This experiment will provide evidence for treatment of UC by targeting the phosphorylation of tight junction protein. As far as I know, this kind of research have not been reported in the literature.

溃疡性结肠炎(UC)是一种结肠粘膜的慢性炎症和溃疡性病变，严重影响生活质量和劳动能力，重症危及患者生命。结肠粘膜屏障功能损伤是UC发生发展的重要环节，紧密连接是结肠粘膜屏障的结构基础。前期实验发现，UC结肠粘膜屏障功能损伤与紧密连接蛋白差异表达有关。文献报道，异常磷酸化导致的紧密连接蛋白变构和极性改变，亦是影响上皮屏障功能的重要因素。我们假设结肠粘膜紧密连接蛋白存在异常磷酸化，并影响到结肠上皮细胞间通透性、细胞损伤/修复、细胞增殖/凋亡，导致结肠粘膜屏障功能损伤。本实验拟通过观察紧密连接蛋白在UC结肠粘膜中的磷酸化状况，并采用过磷酸化、去磷酸化等手段，观察结肠上皮细胞间通透性、细胞损伤/修复、细胞增殖/凋亡的变化，阐述紧密连接蛋白异常磷酸化对结肠粘膜屏障功能的影响及其信号转导机制，为探讨UC发病机制，并为靶向紧密连接蛋白异常磷酸化来治疗UC提供实验依据。本课题研究国内外尚未见相关文献报道。

溃疡性结肠炎(UC)是一种结肠粘膜的慢性炎症和溃疡性病变，严重影响生活质量和劳动能力，重症危及患者生命。结肠粘膜屏障功能损伤是UC发生发展的重要环节，而紧密连接是结肠粘膜屏障的结构基础。本实验结果表明，claudin 1-8、occludin、ZO-1在结肠上皮细胞中均有表达，位于细胞连接之处，其表达强度有所不同，以claudin-3、claudin-7表达较强，occludin、ZO-1、claudin-4表达中等，claudin-1,2,5,6,8表达较弱。细菌脂多糖(LPS)处理的结肠上皮细胞中，claudin-1、claudin-3、claudin-4表达下降，磷酸化claudin-3亦减少；磷酸化claudin-5减少，claudin-5表达无变化；磷酸化claudin-6未受LPS的影响。LPS处理的细胞中，磷酸化claudin-7减少，claudin-7表达下降，claudin-8、occludin表达无变化，ZO-1在24h表达略有上升，72h表达下降。表皮生长因子(EGF)处理的结肠上皮细胞中，claudin-1、claudin-2、claudin-3、claudin-4表达上调，而磷酸化claudin-3减少。磷酸化claudin-5明显增多，claudin-5表达减少；磷酸化claudin-6有所减少。EGF处理的T84细胞中，磷酸化claudin-7增多，claudin-7表达无明显变化，claudin-8、occludin表达上调，ZO-1在24h表达显著上升，72h表达下降。此外， LPS会增加结肠上皮细胞间隙的通透性，增加上皮细胞的凋亡，这一作用可以被磷酸激酶活化剂EGF所阻止。综上所述，某些侵袭因素可以导致紧密连接蛋白磷酸化异常，多数是磷酸化减弱；靶向磷酸化可能减少细胞间隙通透性，减少凋亡，促进细胞迁移，以及加强细胞间紧密连接的稳固。