DON诱导断奶仔猪免疫应激的表观遗传组学机制研究

In modern pig industry, mycotoxin poisoning has become an important factor of early weanling stress syndrome in piglets. Deoxynivalenol (DON) is one of the most serious mycotoxin which severely contaminates grains and feeds in our country. Our preliminary experiments indicated that exposure to DON induced cytokines and chemokines over expression leading to immune stress, which caused immune function damage. Pig is the most sensitive animal to DON. However, there are very few studies working on DON-induced immune stress in pig and the mechanisms remain unclear. In this project, we will focus on spleen which is the target organ of DON to investigate the effects of DON-induced immune stress in the growth and development, physiological and immunological function, amount and subgroup of T cell, proliferation and apoptosis of T cell, level of immunoglobulin, level of cytokine and chemokine, expression of immune-related gene and MAPK signal pathway. Genome-wide DNA methylation, histone modification and microRNA expression profiles are determined in immune stress piglets induced by DON. The key relative genes and epigenetic factors will be determined by bioinformatics analysis. The ultimate goal of these studies is to further reveal mechanisms of DON-induced immune stress in weanling piglets from epigenomics perspective. These findings are expected to offer significant targets and ideas that will discover efficacious interventions to food poisoning of DON over what we have known up to date.

在现代养猪业，霉菌毒素中毒已经成为诱发仔猪早期断奶应激综合征的重要诱因。脱氧雪腐镰刀菌烯醇（DON）是我国污染谷物和饲料最严重的霉菌毒素之一。我们前期研究发现，DON能够诱导细胞因子和趋化因子过表达，引起免疫应激，损害机体免疫功能。猪是对DON最敏感的动物，但是关于DON诱导猪免疫应激的研究极少，机理不清楚。本课题以DON的靶器官脾脏为切入点，研究DON诱导的免疫应激对断奶仔猪生长发育、生理和免疫功能的影响；分析DON对T淋巴细胞数量与亚群、增殖与凋亡、免疫球蛋白、细胞因子与趋化因子、免疫相关基因与MAPK信号通路的影响；以表观遗传组学为体系，在全基因组范围内获取DON诱导断奶仔猪免疫应激的DNA甲基化谱、组蛋白乙酰化谱和miRNA表达谱数据；通过生物信息学分析，确定关键基因和表观因子，深入阐释DON诱导断奶仔猪免疫应激的表观遗传组学机制，为有效防治DON中毒提供新的靶点和思路。

脱氧雪腐镰刀菌烯醇（DON）是一种由镰刀菌产生的B型单端孢霉烯族毒素，是谷物及谷物制品中最常见的真菌毒素之一。DON可以引起人和动物一系列的不适反应，包括恶心、呕吐、拒食、生长抑制等，还可以扰乱神经内分泌系统和免疫系统。然而，DON诱导免疫应激机理尚不完全清楚。本研究从DNA甲基化、miRNA测序（miRNA-seq）着手，探索了DON诱导断奶仔猪免疫应激的表观遗传学机制。DON可引起免疫应激，降低断奶仔猪的生产性能，引起外周血中T淋巴细胞比例、血清中IgG和细胞因子IL-1β、IL-6和TNF-α显著上升。提取仔猪脾脏DNA，进行简化代表性重亚硫酸盐测序文库构建测序，对测序结果进行分析，绘制了染色体Promoter和CGIs甲基化图谱。对差异表达的基因进行GO和KEGG功能富集分析，发现富集差异不显著，但差异表达基因主要涉及细胞分解代谢调节、细胞增殖负调控、小脑发育和有丝分裂细胞周期调控等生物过程。对猪脾脏进行miRNA-seq，绘制了DON诱导免疫应激断奶仔猪脾脏的miRNA图谱，鉴定了差异表达的miRNA，并对这些miRNA的靶基因进行了预测，为深入研究DON诱导断奶仔猪免疫应激的表观遗传学机制提供了新的线索。. 肠上皮是抵抗食物污染物的第一道屏障，对霉菌毒素高度敏感。DON通常会引起炎症并影响人类和动物的肠道健康。本研究使用猪肠上皮细胞系（IPEC-J2）研究DON诱导猪肠炎的潜在机制。DON诱导促炎基因表达，增加了IL1A，IL6和TNF-α的蛋白质水平。DON激活ERK1/2，JNK和p38 MAPK的磷酸化。抑制p38减弱了DON诱导的IL6，TNF-α，CXCL2，CXCL8，IL12A，IL1A，CCL20，CCL4和IL15的产生，抑制ERK1/2部分下调IL15和IL6的水平。且p38抑制剂可降低IL1A，IL6和TNF-α的蛋白水平的上调，ERK1/2抑制剂部分减弱IL6的蛋白水平的上调。DON通过激活p38和ERK1/2诱导IPEC-J2细胞中促炎因子的产生。