RIG-I在维生素A 调节儿童肠外营养相关性肠粘膜免疫功能损伤中的作用机制研究

Total parenteral nutrition is an essential method of nutritional support for critically ill pediatric patients unable to feed enterally. Unfortunately, long-term use of total parenteral nutrition may be associated with impairment of gut mucosal immunity. The gut mucosal immunity is regulated by dendritic cell. It has been demonstrated that total parenteral nutrition could mediate the activation of dendritic cell, and interfere with the production of secretory immunoglobulin A (sIgA). Our recent studies have revealed that the concentration of serum vitamin A was positively related to sIgA levels in stool samples in pediatric patients with total parenteral nutrition. These results suggest that vitamin A status may influence the gut mucosal immunity of pediatric patients receiving total parenteral nutrition. However, the exact mechanism requires further elucidation. Retinoic acid inducible gene-I (RIG-I) was originally identified as the gene induced by retinoic acid - the active metabolites of vitamin A, and was considered to participate in the regulation of the immune response. Thus, we hypothesize that RIG-I signaling may involved in the molecular mechanism of vitamin A for modulating gut mucosal immunity. This project proposes to create an animal model of total parenteral nutrition, analyze the changes of immune function markers following vitamin A intervention. In addition, the dendritic cells are isolated from gut mucosal and cultured in vitro, and the RNA interference technology is used to create different models of RIG-I gene expression. After vitamin A intervention, the functional differentiation of dendritic cells and the variable expression of signaling molecules in RIG-I mediated pathway are compared among different models. Our study will elucidate the mechanism by which vitamin A modulate the total parenteral nutrition-related gut mucosal injuries through RIG-I signaling pathway. These issues could not only provide evidence for rational use of vitamin A to improve gut immunity but also provide theoretical basis for preventing total parenteral nutrition-related gut mucosal injuries in children.

肠外营养是危重患儿营养支持的必要手段，然而长期应用肠外营养可损害肠粘膜免疫功能。肠粘膜免疫受树突状细胞（DC）调控，肠外营养已被证实可介导DC活化，干扰sIgA产生。我们前期研究发现，肠外营养儿童血清维生素A与粪便sIgA水平正性相关，提示维生素A可影响肠外营养儿童肠粘膜免疫力，然而其确切机制还有待进一步阐明。视黄酸诱导基因-I（RIG-I）参与机体免疫反应，受维生素A活性产物-视黄酸调节，可能是维生素A调节肠粘膜免疫力的分子途径。本项目建立肠外营养动物模型，分析维生素A干预下肠粘膜免疫指标的变化；同时分离肠粘膜DC体外培养，通过RNAi干扰构建不同RIG-I表达模型，视黄酸干预后比较不同模型中DC分化水平及RIG-I信号通路相关分子表达变化，阐明RIG-I在维生素A调节肠外营养相关性肠粘膜损伤中的作用机制，为临床合理使用维生素A改善肠道免疫力，防治肠外营养相关性并发症提供理论依据。

肠外营养是危重患者营养支持的必要手段，然而长期应用肠外营养可损害肠粘膜免疫功能。本项目的前期研究表明：维生素A营养状态可调节肠外营养患者的肠粘膜免疫功能，然而，其确切的分子机制还需要进一步研究证实。视黄酸诱导基因-I（Retinoic acid inducible gene-I，RIG-I）参与机体免疫反应，受维生素A活性产物-视黄酸调节，可能是维生素A改善肠粘膜免疫力的分子途径。本项目在原有工作基础上，以肠外营养相关性肠粘膜免疫损伤为切入点，通过动物实验和体外细胞培养，研究RIG-I在维生素A调节免疫细胞分化，改善肠粘膜免疫功能中的作用机制。. 本研究建立SD大鼠肠外营养模型，分离肠粘膜树突状细胞，给予维生素A干预，完成各干预组肠粘膜免疫相关指标的检测及RIG-I信号通路相关分子mRNA与蛋白表达水平的分析，结果显示：1肠外营养组SD大鼠肠粘膜树突状细胞的数目与对照组相比无明显差异，但CD80和CCR7的阳性面积显著高于对照组（P<0.01），说明介导树突状细胞的分化成熟，是肠外营养损害肠粘膜免疫功能的可能途径之一。体外细胞培养结果显示：培养48小时，视黄酸干预组不成熟树突状细胞百分比增高，提示视黄酸（维生素A活性产物）干预可抑制树突状细胞分化成熟。2与对照组相比，细胞培养上清中促进sIgA分泌的细胞因子IL-6，IL-10水平显著增高（P<0.05）。提示视黄酸干预可影响树突状细胞产生促进sIgA 分泌的细胞因子的表达，有助于sIgA 的分泌。3肠外营养干预下SD大鼠肠粘膜TLR2，TLR4和MyD88的表达均低于对照组（P<0.01）。提示肠外营养干预使粘膜抗原识别受体及其适配器蛋白表达下调，可能降低肠粘膜的抗原识别能力，进而导致肠道免疫力低下。4肠外营养干预可下调SD大鼠肠粘膜树突状细胞中RIG-I通路中部分关键基因的表达；而补充视黄酸可上调这些基因的表达，可能是其促进肠粘膜免疫保护的作用机制。 . 本研究在分子层面揭示了RIG-I是维生素A发挥肠粘膜免疫调节作用的关键因素，为临床合理使用维生素A促进肠粘膜免疫力，防治肠外营养相关性肠道并发症提供新的理论和实验依据。