鸭GRα基因对小肠黏膜高温应激损伤的影响及GC参与的保护机制研究

The samll intestinal mucosal damage caused by heat stress impacts its absorption function and induces a variety of diseases, which is the huge challenge faced by the scale livestock and poultry raising industry in the summer, and then the molecular mechanism of intestinal mucosal damage is got more and more attention. In order to improve the body's resistance, adrenal cortexs secrete a mass of GC when animals are subjected to harmful stress. GRα, the executor of the effects of GC, plays an important role in life activities. However, the functional mechanism of GRα gene protecting intestinal mucosa from damage induced by stress is not well understood. In this project, the expression and location of GRα gene will be investigated in the small intestinal mucosa of duck suffered by different time of heat stress. The relationship between the expression of GRα gene and the changes in shape and structure of small intestinal mucosa epithelial cells will be revealed. By constructing cell lines with high/RNAi expression of GRα gene, the signaling pathways regulated by GRα gene and involved in anti-apoptosis in duck samll intestinal epithelial cells treated by heat stress will be isolated and validated. By building duck mutants with high/interference expression of GRα gene in small intestine, the effect of its expression in small intestinal mucosa injury induced by heat stress will be defined. The protecting mechanism of the effects of GRα gene in small intestinal mucosa damage induced by heat stress would be illuminated. The results of this research will not only provide a scientific basis for alleviating animal damage caused by heat stress, but also open a new train to investigate the mechanism of intestinal mucosa damage in the medicine.

高温应激导致小肠黏膜损伤，影响其吸收功能，诱发各种疾病，是规模化畜禽养殖业在夏季面临的巨大难题，因此，肠黏膜损伤机制的研究也越来越引起人们的重视。当动物遭受有害应激时，肾上腺皮质分泌大量GC，以提高机体的抵抗力，而GRα作为GC效应的执行者，在生命活动中具有重要作用，但GRα基因对肠黏膜应激损伤的保护机制研究尚属空白。本项目拟对GRα基因在不同时间高温应激鸭小肠黏膜中的表达和定位进行研究，揭示该基因表达水平与小肠上皮细胞形态结构变化间的关系。通过构建GRα基因高/抑制表达细胞系，筛选并验证受其调控的参与抑制高温应激上皮细胞凋亡的信号通路。通过构建鸭小肠黏膜GRα基因高/干扰表达的突变体，明确该基因对肠黏膜高温应激损伤的影响，阐明GRα基因对肠黏膜高温应激损伤的保护作用机理，本研究结果有望可以为缓解动物高温应激损伤提供科学依据，同时也为医学上肠黏膜损伤机制的研究开辟新的思路。

高温应激在现在的集约化养殖模式中已成为常见的应激原之一，而动物的小肠黏膜在经受高温应激时十分容易受到损伤，导致其机体代谢和生产性能受到严重的不良影响，目前小肠黏膜应激损伤的分子机制的研究越来越受到重视。GRα是GC效应的执行者，在生命活动中具有重要作用。本项目以GRα基因为研究目标，首先探究了在模拟南方地区夏季高温环境（9:00-15:00，26-41℃循环变温）的应激条件下，不同时间点（1-15天）鸭的小肠形态学结构以及血液生化指标，其中在高温应激第7天时，小肠的形态学结构和指标都发生了显著下降，在第3-10天范围内，血清指标出现显著差异，其中皮质醇、甲状腺素、内毒素和补体C4的都有升高的趋势，谷丙转氨酶则总体呈现降低趋势。随后对高温应激第15天的鸭十二指肠、空肠和回肠进行了差异转录组学分析，与实验对照组相比，分析得到了十二指肠中的615个差异表达基因，空肠中的285个差异表达基因以及回肠中的89个差异表达基因。本研究成功构建了包装干扰GRα基因过表达和抑制表达RNA的慢病毒，在鸭小肠上皮细胞中验证了其干扰效果，随后在活体上构建出鸭肠黏膜GRα基因高表达和抑制表达的突变个体，通过对其进行同样条件的高温应激饲养，初步表明GRα基因的表达水平与小肠结构的高温应激损伤存在一定程度的关联，GRα基因过表达实验组的十二指肠、空肠和回肠绒毛结构损伤程度最低，而高温应激对照组和GRα基因抑制表达组的三个肠段结构损伤依次增大。证实了GRα基因对鸭小肠黏膜高温应激损伤的保护作用相关，且提高GRα基因的表达水平能够在一定程度上减少小肠结构的热应激损伤，本研究结果不仅为缓解动物高温应激损伤提供了科学依据，同时也能够开辟医学上肠黏膜损伤机制研究的新思路。