MiR-155靶向调控claudin-1对脑损伤后肠黏膜屏障功能的影响及机制研究

Traumatic Brain injury(TBI) patients are often associated with intestinal mucosal dysfunction, and claudin-1 plays an important role in the function of the intestinal mucosa. Our previous work has shown that miR-155 was more highly expressed in peripheral blood of patients with traumatic brain injury compared with control group. We also found an increased expression of miR-155 and a decreased expression of claudin-1 in mouse intestinal mucosa with TBI. We clouded that miR-155 may regulate intestinal epithelial tight junction by adjust the template mRNA of claudin-1. Further validation of the adjustment involved the dual luciferase reporter pull-down assay system. qPCR and Western blot were used to detect the expression of miR-155 and claudin-1. Plasma endotoxin, diamine oxidase, D-lactic acid and intestinal epithelial barrier function in animal models and patients will be observed in order to get convincing results in vivo. The aim of this study was to explore the epigenetic regulation of the intestinal mucosal barrier, and also, we expect that miR-155 will be a new clinical diagnostic marker in patients with intestinal mucosal dysfunction after TBI or other traumatic disease.

颅脑外伤患者常合并肠黏膜功能障碍，claudin-1在肠黏膜功能中起着重要作用。我们前期工作表明，颅脑外伤患者外周血中miR-155表达增高，动物实验也提示颅脑损伤小鼠肠道黏膜中miR-155表达增高同时claudin-1表达下降。我们推测miR-155可能通过靶向调控claudin-1对肠黏膜屏障功能进行调节。本研究拟通过qPCR、蛋白印迹等方法检测miR-155对claudin-1调节的效果；通过双荧光素酶报告基因检测系统和Pull-down实验确认miR-155对claudin-1的调控的分子机制；建立小鼠颅脑损伤模型检测miR-155、claudin-1及肠黏膜屏障功能之间的关系；观察动物模型和临床患者miR-155与内毒素、二胺氧化酶、D-乳酸以及肠上皮细胞屏障功能相关性。本研究旨在明确颅脑损伤后肠黏膜屏障功能障碍分子机制及病理改变，为发现新的临床诊断标志物和治疗方法提供实验依据

颅脑外伤患者常合并肠黏膜功能障碍，claudin-1在肠黏膜功能中起着重要作用。但目前对于各种创伤或应急状态下后肠道claudin-1 蛋白表达调控机制并不十分明确。本项目在前期工作基础上，通过建立小鼠颅脑损伤模型，观察动物模型和临床患者miR-155与血浆内毒素、二胺氧化酶、D-乳酸以及肠上皮细胞屏障功能相关性，探讨了miR-155、claudin-1及肠黏膜屏障功能之间的关系，明确miR-155对claudin-1调控的分子机制；确认了肠道组织和血浆中miRNA-155的改变与颅脑损伤后肠道粘膜屏障功能障碍密切相关；颅脑损伤后肠道粘膜屏障功能障碍是通过miRNA-155的高表达来介导的。研究发现：TBI患者伤后24 h外周血miR-155基因表达升高，而外周血claudin-1的表达下降，提示miR-155可能引起肠道claudin-1蛋白含量的下调，促使上皮紧密连接复合体降解，进而引起肠黏膜损伤；通过ELISA检测到TBI患者伤后24 h 肠黏膜损伤的标志物DAO和I-FABP有不同程度的升高；在动物实验中，以AAV-DJ为载体，下调miR-155可提高TBI后7d、14d小鼠claudin-1的表达，并减轻肠黏膜损伤的程度；以慢病毒为载体，上调miR-155可抑制Caco-2细胞claudin-1 mRNA和蛋白质的表达，而抑制miR-155可上调Caco-2细胞laudin-1 mRNA和蛋白质的表达，上调和下调miR-155对occludin、ZO-1、MLCK等肠上皮蛋白基因表达无明显的影响。上述结果为血浆中miR-155作为颅脑损伤后评估患者肠道屏障功能损伤的生物学标志物提供了理论依据。