脓毒症发生中miR-204参与调控单核巨噬细胞糖皮质激素应答的分子机制研究

Immunosuppression is a key point in the development of infection induced by serious trauma to sepsis. Our previous studies showed that the functions of macrophages could be regulated by the pathway of "corticosteroids (GCs) -corticosteroid receptor (GR) -endoplasmic reticulum", but the upstream mechanism is still unclear. Our preliminary experiment find out the differential expression of miRNAs molecular using miRNA chip screening out in sepsis process. During such miRNAs, The blocked miR-204 can significantly reduce the immune suppression of GC on macrophages. Forecast analysis suggests GRαmay be miR-204 target, thus speculate: miR-204 may regulate macrophages GCs response through regulating GRα/GR β expression proportion, which participate in sepsis immunosuppression formation. Focusing on mononuclear cell/ macrophages for target, this study is intended to confirm the direct regulation of miR-204 on GRα, and clarify the relationship between miR-204α control way and mononuclear cell/ macrophages' glucocorticoid response, by using the method such as the designated mutations, miRNA genetic target report. And then explore the functions and molecular mechanism of miR-204 in GCs regulation on mononuclear cell/ macrophages. The results will deepen our understanding of sepsis pathogenesis, and will provide new targets for clinical intervention to serious injuries and immune disfunction patients.

免疫抑制是严重创伤后感染发展成脓毒症的关键机制。我们前期研究发现糖皮质激素(GCs)作用于其受体(GR)后可通过内质网应激调控巨噬细胞功能，但其上游机制不清，预实验采用miRNA芯片筛查出在脓毒症过程中差异表达的miRNAs分子，其中miR-204被阻断后可显著增强GCs对巨噬细胞的免疫抑制效应，预测分析提示GRα可能是miR-204的靶标，由此推测：miR-204可能通过调控GRα/GRβ表达比例来调控单核巨噬细胞GCs应答反应，参与脓毒症免疫抑制的形成。本研究拟以单核巨噬细胞为对象，采用定点突变、miRNA 靶标报告基因检测等方法证实miR-204对GRα表达的直接调控作用，并明确miR-204-GRα调控途径与单核巨噬细胞糖皮质激素应答间的内在联系，从而阐明miR-204在GCs 对单核巨噬细胞免疫调控中的作用及分子机制。研究结果将深化对脓毒症发病机制的认识，为临床干预严重创伤免疫紊

免疫抑制是严重创伤后感染发展成脓毒症的关键机制。我们前期研究发现糖皮质激素(GCs)作用于其受体(GR)后可通过内质网应激调控巨噬细胞功能，但其上游机制不清，预实验采用miRNA芯片筛查出在脓毒症过程中差异表达的miRNAs分子，其中miR-204 被阻断后可显著增强GCs 对巨噬细胞的免疫抑制效应，预测分析提示GRα可能是miR-204 的靶标，由此推测：miR-204 可能通过调控GRα/GRβ表达比例来调控单核巨噬细胞GCs 应答反应，参与脓毒症免疫抑制的形成。本研究收集了临床脓毒症患者不同时间点外周血，检测了其单核细胞比例、单核细胞miR-204/GR 表达和免疫功能、外周血皮质酮水平，并分析了免疫功能抑制前后单核细胞miR-204/GR 表达与皮质酮水平之间的关系，发现随免疫抑制程度的加深，其皮质醇浓度有相应增高趋势，单核细胞miR-204表达逐渐增高；利用体外单核巨噬细胞模型，检测了miR-204对GRα表达的调控作用，免疫印迹证实过表达miR-204 直接抑制巨噬细胞GRα表达，而阻断miR-204 直接促进巨噬细胞GRα表达，LPS+皮质酮处理后GCs通过miR-204参与对GRα/ GRβ蛋白表达比例的调控，初步证明GCs是通过GRα途径来实现对巨噬细胞免疫调控作用这一重要分子机制。研究结果将深化对脓毒症发病机制的认识，为临床干预严重创伤免疫紊提供实验依据。