miR-199a对断奶仔猪肠道巨噬细胞极化的影响及其机理研究

Weaning stress in piglets impairs intestinal mucosal barrier and immune function. It has been reported that M1 and M2 macrophage polarization plays a pivotal role in maintaining immune homeostasis. Our previous results show that weaning stress down-regulates intestinal miR-199a expression and many predicted target genes of miR-199a are involved in the regulation of macrophage polarization. It is thus reasonable to speculate that miR-199a may manipulate macrophage polarization in piglets and thereby alleviate weaning stress. This project intends to study the effect of miR-199a on macrophage polarization by RT-PCR and Western Blot technology. Flow cytometry and IPEC-J2 wound-healing model will be used to evaluate the phagocytic activity and wound-healing function of polarized macrophage. Luciferase reporter assay and siRNAs will be used to identify the targets of miR-199a. Finally, DSS colitis model will be used to confirm the effect of miR-199a on macrophage polarization in miR-199a-/- mice. Together, these data may clarify the mechanism of miR-199a-mediated regulation of macrophage polarization and provide valuable evidence for the potential use of miRNA as new immune-modulatory agent in regulation of weaning stress.

断奶应激会造成仔猪肠黏膜损伤，导致仔猪免疫机能紊乱。巨噬细胞通过其不同的极化表型（M1和M2）维持肠道免疫系统的稳态。研究发现断奶应激下调miR-199a表达，而靶基因预测表明miR-199a对巨噬细胞极化相关信号通路具有关键调控作用。因此，有理由推测miR-199a可能通过影响肠道巨噬细胞极化状态进而调控仔猪的断奶应激。本项目拟以仔猪肠道巨噬细胞为研究对象，运用RT-PCR和WesternBlot等技术研究miR-199a对巨噬细胞极化的影响；运用流式细胞术和肠上皮细胞损伤模型，探讨miR-199a对巨噬细胞吞噬杀菌和肠道修复功能的影响；运用双荧光素酶报告基因和siRNA技术验证miR-199a的靶向基因，最后利用miR-199a敲除小鼠DSS结肠炎模型进行结论验证。研究结果将初步阐明miR-199a对仔猪肠道巨噬细胞极化的影响及其分子机制，为仔猪断奶应激调控提供理论依据和新思路。

断奶应激会造成仔猪肠黏膜损伤，导致仔猪免疫机能紊乱。巨噬细胞通过其不同的极化表型（M1和M2）维持肠道免疫系统的稳态。研究发现断奶应激下调miR-199a表达，而靶基因预测表明miR-199a对巨噬细胞极化相关信号通路具有关键调控作用，但miR-199a对巨噬细胞极化状态的影响以及对肠道炎症的调控机制尚不清楚。本研究运用了RT-PCR和转录组学测序等技术发现miR-199a能促进M1而抑制M2型巨噬细胞极化；进一步利用miR-199a敲除小鼠DSS结肠炎模型，分析了miR-199a对肠道发育以及肠道炎症的影响；最后运用双荧光素酶报告基因技术验证了miR-199a的靶向基因，发现NDRG1可能作为潜在靶基因参与巨噬细胞极化及肠道炎症的调控过程。因此，通过本项目的实施，初步揭示了miR-199a对巨噬细胞极化及肠道炎症的影响及其分子机制，为仔猪断奶应激调控提供了理论依据和新思路。