Syndecan-1调控紧密连接在肠上皮屏障维护中的作用及机制研究

Gut barrier impairment is the initiator of many local and systematic immune or inflammatory diseases. The intestinal epithelial tight junction is considered as the principal determinant of gut barrier function. Howover, isolated defects in tight junction are insufficient to cause diseases, which suggesting that some other factors besides the junction may participate in the maintainance of intestinal epithelial barrier function. Emerging evidences of syndecan-1(Sdc1), the predominant cell surface heparin sulfate proteoglycan, in maintaining murine and human intestinal epithelial barrier function has drawn our attention. Our previous studies had demonstrated that Sdc1 could alleviate the impairment of intestinal barrier, increased mucosal permeability, bacterial translocation and even sepsis induced by inflammatory stimuli. Meanwhile, Sdc1 could upregulate the expressions of tight junction proteins such as occludin and ZO-1. And defected tight junction function could also be repaired by Sdc1. However, how Sdc1 protects gut barrier fucntion is unkown. The hypothesis driving this proposal is that Sdc1 maintains the gut barrier function through regulating tight junction express and assembling. In this proposal, lenti-virus construct, RNAi and trunction or site-directed mutagenesis were used to regulate Sdc1 expression and Sdc1 constructs were transfected epithelial cells and barrier function deficient mice. The interactions of Sdc1 and tight junction proteins,involved signalling pathways and their effects on intestinal barrier funtions were observed. The aim of this proposal is to elucidate how Sdc1 reguates tight junctions in maintaining intestinal epithelial barrier fuction and to find out the exact interation sites of Sdc1. Our study may develop Sdc1 as a new therapuetic target for intestinal barrier dysfunction.

肠上皮屏障损伤是导致肠道乃至全身多种炎症/免疫性疾病的重要启动环节。尽管上皮间紧密连接(TJ)被认为是肠上皮屏障的结构基础，但单纯TJ缺陷并不足以致病，表明肠上皮屏障存在其它保护性机制。我们前期已发现硫酸肝素蛋白聚糖Syndecan-1(Sdc1)能明显减轻炎症刺激后的上皮屏障损伤、通透性增加、细菌易位甚至脓毒症的发生，且Sdc1能上调TJ蛋白表达并修复TJ缺陷细胞的屏障功能受损，由此我们提出肠上皮屏障一个新的保护性机制：Sdc1通过调控TJ的表达和结构，协同维护肠上皮屏障功能。本项目拟进一步采用慢病毒载体构建和RNAi干扰技术调节Sdc1的表达，在单层上皮屏障模型和屏障功能缺陷小鼠中观察Sdc1对TJ及其调控信号通路、上皮屏障结构和功能的影响，并通过截短突变/点突变技术寻找Sdc1与TJ作用的位点，旨在阐明Sdc1对肠上皮屏障的保护机制，为防治粘膜屏障失调相关疾病提供新的干预途径。

肠上皮屏障损伤是导致肠道乃至全身多种炎症/免疫性疾病的重要启动环节。尽管上皮间紧密连接（Tight junction, TJ）被认为是肠上皮屏障的结构基础，但单纯TJ缺陷并不足以致病，表明肠上皮屏障存在其它保护性机制。在本项目中，我们证实了肠上皮屏障一个新的保护性机制：Sdc1通过调控TJ的表达和结构，协同维护肠上皮屏障功能。本项目主要采用慢病毒载体构建和RNAi干扰技术调节Sdc1的表达，在单层上皮屏障模型和屏障功能缺陷小鼠中观察Sdc1对TJ及其调控信号通路、上皮屏障结构和功能的影响，并通过截短突变/点突变技术寻找Sdc1与TJ作用的位点。我们发现Sdc1通过调控TJ的表达和结构，协同维护肠上皮屏障功能。Sdcl对肠上皮屏障的维护具有保护作用，表现为能够显著稳定上皮的完整性、抑制细菌易位和降低跨膜转运的通透性。在细菌、毒素、炎症因子等损伤因素刺激下，Sdc1一方面通过胞内段直接与TJ蛋白或细胞骨架作用，稳固TJ复合物的结构，另一方面Sdc1能够激活Stat3信号通路，磷酸化的Stat3直接结合Z0-1和occludin的启动子区域，调控ZO-1和occludin的转录，从而参与Sdc1对屏障功能的维护；Z0-1与occludin具有相互作用，构成免疫复合体，能负反馈调节 Sdc1表达，共同构成环路维护屏障功能保持正常。一旦Sdc1对TJ的上述调控机制被破坏，Sdc1将失去对TJ受损的修复机制，上皮屏障损伤持续加重，肠壁通透性增加，细菌易位加剧，引发或加重炎症性肠病。.我们进一步发现UC患者肠上皮Sdcl的表达明显减少，血清中游离Sdcl水平明显增加，且血清中Sdcl水平与升高的中性粒细胞数目呈正相关。细胞实验表明Sdc1胞外段脱落伴随着炎性因子分泌的增多及NF-KB通路的激活，抑制Sdcl脱落可明显减少炎性因子的分泌及NF-KB通路的激活。在DSS诱导的结肠炎小鼠模型中，抑制Sdcl脱落可减轻组织学损伤、减少细胞因子的分泌及NF-KB通路的激活，且脱落的Sdc1与中性粒细胞募集相关。