APS调控种公鸡Th细胞因子表达的父系传代表观遗传机制

Broiler chickens exposured to a large number of stressors during the growth process of high-intensity, which would impair immune system function and led to various conditional diseases. It is becoming increasingly clear that Key nutrients can modulate not only the organisms directly exposed but also their offspring, even though such factors do not cause DNA mutations. So, nutriepigenetics will provide fresh insights into the mechanisms underlying the induction and propagation of transgeneration properties in immune function modulated by nutrients. Four tightly integrated trials will be conducted to investigate the paternally induced transgeneration epigenetics modulation of APS on cytokines gene expression of Th lymphocytes in chickens. Firstly, the Th cytokine expression characteristics will be measured by the specific gene chip for immune effector molecule in male breeders and their offspring in order to get the specifc cytokines possessing paternally induced transgeneration epigenetics modulation of APS. Secondly, effects of APS exposure of male breeders on the chromatin structure and the position of Th cytokine genes in the nucleus of the Th cytokine loci in male breeders and their offspring will be analyzed by DnaseI / qPCR and FISH, which will provide some informations about pericentromeric localization of Th cytokine loci. Thirdly, DNA methylation at the Th cytokine promoter/enhancer/silencer in male breeders and their offspring will be analyzed by Epityper methylation assay. The last not the least, histone modifications (H3K4, of H3K9 and H3K27) and transcription factor binding at the Th cytokine promoter/enhancer/silencer in male breeders and their offspring will be anzlyzed by ChIP/qPCR assays. The above epigenetic parameters in male breeders and their offspring will enable us to perform the first in-depth study of the induction, inheritance and repair of a poultry transgeneration epigenetics, with general implications and theoretical support for transgeneration immune modulation by nutriepigenetics by key nutrients, such as functional polysaccharides.

肉鸡在高强度的生长过程中会接触大量应激原，使得机体免疫系统功能异常，导致各种条件性疾病频发。营养表观遗传学的研究为关键营养素传代性调控机体免疫功能提供了全新的研究视角。本项目利用特异性免疫效应分子基因芯片探寻黄芪多糖（APS）调控肉种公鸡Th细胞因子表达特性，经分析种公鸡及其商品代肉鸡细胞因子表达特点研究APS的免疫调控作用的可传代性；利用FISH和DnaseI/qPCR技术分析APS调控种公鸡及其后代Th细胞因子基因位点染色质三维空间结构特性；采用DNA甲基化技术研究APS调控种公鸡及其后代Th细胞因子表达顺式调控元件变化规律；经ChIP/qPCR技术研究APS调控种公鸡及其后代Th细胞因子结合组蛋白修饰特性。旨在阐明APS调控肉种公鸡Th细胞因子传代表达的营养表观遗传机制，为肉鸡具有可传代性免疫调控功效多糖的目的性筛选、营养表观遗传关键营养素的开发研究提供理论支撑。

肉鸡遗传选育改善偏重肉鸡生长性能致免疫性能削弱。本课题探索种公鸡日粮添加黄芪多糖（Astragalus polysaccharides，APS）对商品代系统免疫和肠道黏膜免疫的影响及APS传代调控效应的表观遗传机制。种公鸡日粮中添加APS（0，0.01，0.1，1，10 g/kg）并进行孵化试验获得商品代肉鸡构建传代模型，所取得的研究结果如下：.1. 种公鸡日粮添加10 g/kg 的APS可通过提高紧密连接蛋白的表达保证肠道黏膜上皮的完整性； 10 g/kg APS显著提高了种公鸡空肠绒毛高度和绒毛高度隐窝深度比值，降低了14日龄时肉仔鸡空肠隐窝深度，提高空肠绒毛高度隐窝深度比，此调控效应具有传代性，改善了商品代肉仔鸡前期的生长性能。.2. 种公鸡日粮添加APS可通过影响胸腺组织中细胞骨架相关基因表达，抑制种公鸡胸腺组织退化；通过影响MHC的表达影响机体的免疫耐受状态，从而调控种公鸡和商品代肉仔鸡胸腺的免疫性能，检验所筛选目的基因MHC-BF-1启动子DNA甲基化修饰发现在ASP传代调控胸腺基因表达过程中存在DNA甲基化修饰的调控作用。.3. 种公鸡日粮添加APS可传代调控商品代肉鸡脾脏TLR4信号通路活性，产生内毒素耐受样的免疫反应，但对免疫耐受相关调控因子的表达无影响；检验脾脏组织MyD88和TRIF基因启动子区域的DNA甲基化修饰状态，发现存在其修饰调控效应。.4. 种公鸡日粮添加APS可影响商品代肉仔鸡的肠道黏膜免疫状态，产生内毒素耐受样免疫反应， APS可激活TLR4信号通路下游病原清除相关的TRIF信号通路，这种内毒素耐受样免疫反应由SOCS1和STAT1诱导的下游信号通路激活调控。.5. APS可诱导种公鸡睾丸中33个miRNA的差异表达，miRNA靶基因预测和功能分析发现APS可影响睾丸免疫相关“自然杀伤细胞介导的细胞毒性作用”信号通路，gga-miR-16与此通路密切相关。APS可通过下调miR-16-5p的表达进而上调IFNGR2和MAP2K1基因的表达，调节信号通路活性。.综上所述，种公鸡日粮添加APS可发挥免疫和物质代谢的传代调控效应，此效应源于APS在肠道黏膜免疫产生的内毒素耐受样调控效应，进而影响肉鸡系统免疫功能状态，miRNA、DNA甲基化等表观遗传机制在其传代过程中具有一定调控效应。

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