Par-3通过myosin II调控TJ蛋白转运对肠屏障功能的作用及机制研究

Tight junction(TJ) plays a critical role in the maintenance of intestinal barrier function(IBF). The destruction of TJ is not only due to the altered expression of TJ proteins, but also due to the abnormally membrane trafficking of TJ proteins. So far, the regulatory mechanism of membrane trafficking have not been fully elucidated. Recently studies and our previous work have demonstrated that Par-3 may be involved in the membrane trafficking of TJ proteins in intestinal epithelial cells. Meanwhile, Par-3 can control the function of myosin II. Recent data also has revealed a requirement for myosin II in the migration of transport carriers that formed at the trans Golgi network (TGN) and were destined for the cell surface. We hypothesize that the Par-3 may disturb Golgi-membrane trafficking of TJ proteins through regulation of myosin II. Therefore, this study will investigate the role and regulatory mechanism of altered Golgi-membrane trafficking of TJ proteins in the loss of IBF both in vivo and in vitro models, utilizing RNA interference, laser confocal microscopy technique and so on. Thus, this study will inaugurate a new way for the research of IBF injury mechanism and its prevention.

紧密连接(TJ)在维持肠黏膜屏障功能(IBF)的稳定中具有重要作用。多种损伤因素不仅能引起TJ蛋白的表达变化，还能影响其在细胞内的转运过程，从而引起TJ的损伤及IBF的损害。然而造成TJ蛋白转运异常的调控机制尚不明确。新近研究及申请者前期工作发现，细胞极性蛋白Par-3可能参与调控肠上皮细胞TJ蛋白的膜转运；同时，Par-3可调控肌球蛋白myosin II的变化。而myosin II参与转运载体从高尔基体向细胞膜迁移这一过程。因此我们推测：多种病理刺激可引起肠道上皮Par-3的改变，进一步通过调控myosin II，干扰TJ蛋白从高尔基体向细胞膜的转运，破坏紧密连接的形成，继而影响IBF的稳定性。为此，本研究拟采用体内和体外IBF 受损模型，利用免疫荧光、过表达及RNA干扰等技术，阐明Par-3通过myosin II调控TJ蛋白转运对IBF的影响，为IBF的损伤防治提供新的研究思路及靶点。

肠上皮细胞（IEC）以及其完整的紧密连接（TJs）在外部环境和宿主之间形成屏障结构。肠上皮屏障是抵抗存在于肠腔中的毒素和病原体的重要屏障。此屏障的破坏导致肠腔内的毒性抗原向固有层渗透，引发炎症级联反应，促进肠道炎症以及多种肠道疾病的发展，包括炎症性肠病(IBD)。因此深入研究肠屏障的调节机制，有助于为IBD的防治提供新的研究思路及靶点。Par3/Par6/aPKC作为极性蛋白复合物，对肠上皮屏障的调控具有关键作用。本课题围绕Par3/Par6/aPKC复合物对肠屏障的调控作用及机制进行了较为深入的研究。主要结果如下：.1..构建DSS诱导小鼠肠炎模型以及TNF诱导的Caco-2细胞炎症模型，免疫荧光以及WB实验结果显示：体内外肠炎模型中Par-3均表达下降；使用siRNA干扰Par-3的表达，免疫荧光显示：紧密连接蛋白ZO-1的转运不受影响，而occludin蛋白被滞留在高尔基体，其高尔基体-细胞膜转运受到抑制；使用siRNA干扰Par-3的表达，WB检测发现MLCK/MLC/pMLC信号通路活化；而过表达Par-3则显著抑制MLCK/MLC/pMLC信号通路的活化。.2..给予DSS诱导小鼠FICZ（AhR活化剂）活化AhR，可显著缓解DSS诱导的小鼠结肠炎症，并抑制DSS诱导的肠道渗透性增加；AhR活化通过调控TJ结构而不是蛋白水平来抑制TNF-α/IFN-γ诱导的肠上皮屏障损伤；TNF-α/IFN-γ处理细胞以及DSS诱导肠上皮中均可观察到MLCK/MLC/pMLC信号通路显著活化，给予FICZ处理后，MLCK/MLC/pMLC信号通路活化被抑制；AhR通过阻断NF-kB p65信号通路来抑制MLCK/MLC/pMLC的活化。.3..AhR可调节DSS诱导小鼠模型结肠上皮中par3/par6/apkc的表达水平；免疫荧光结果显示过表达par6后，ZO-1结构显著破坏；AhR活化抑制LPS诱导的par6上调导致的肠上皮屏障破坏；Ap-2γ参与AhR活化对par6的调控机制。