【作者】 王宁；

【导师】 戴汉川；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2013， 硕士

【摘要】 随着现代养猪业的快速发展,仔猪的早期断奶已经成为国内外集约化养猪生产的关键技术。然而,断奶仔猪由于免疫系统发育不完善,容易受饲养环境中各种病原体的侵袭,导致免疫应激,使用于生长和骨骼肌蛋白质沉积的养分转而用于维持机体免疫应答,导致其代谢行为发生改变,进而引起生长发育缓慢和生产效率降低,给畜牧业带来严重经济损失,直接影响到猪场的生产水平和经济利益。免疫应激严重影响胸腺的发育,胸腺做为中枢免疫器官在免疫调控中发挥重要作用,目前的研究主要集中在蛋白质编码基因,miRNA介导的转录后水平为免疫应激分子机制提供了新的视角。本研究通过建立断奶仔猪的免疫应激模型,从胸腺中筛选相关差异表达miRNA,并对其进行鉴定和生物信息分析,其研究结果有利于进一步阐明断奶仔猪免疫应激的分子机制,也为利用miRNA及其靶基因信息来降低断奶仔猪免疫应激提供新的思路。本研究主要结果如下：1.将6头大约克夏断奶仔猪(21天断奶)按胎次相同、出生体重相近、随机分为试验组和对照组,每个组别各3头猪。试验组注射100μg/kg BW的LPS,对照组注射等量的生理盐水,3h后,采集断奶仔猪胸腺及其他各种组织。检测试验组和对照组仔猪血浆中白细胞介素-2(IL-2)、白细胞介素-6(IL-6)、前列腺素(PGE2)及生长激素(GH)的含量发现,试验组免疫应激较对照组血浆中炎性细胞因子(IL-2、 IL-6、PGE2)的分泌量增加,而生长激素分泌量显著下降。这说明经过LPS刺激后,试验组断奶仔猪产生了急性免疫应激,发生炎症反应。2.在对试验组和对照组仔猪胸腺总RNA进行提取之后,我们采用Solexa测序技术对试验组和对照组的miRNA文库进行了测序,结果分别获得了高达14.5和13.3百万条短序列读数。通过Solexa深度测序对照组得到126个新miRNA,实验组得到149个新miRNA。Solexa测序得到的对照组和试验组的共15个表达差异显著的miRNA,利用qPCR进行验证,结果与Solexa测序结果一致。对于可能在免疫应激中起重要作用的2个miRNA进行组织表达谱构建。3.将验证后的miRNA进行靶基因预测以及WEGO功能分析发现,ssc-miR-129a、 ssc-miR-1271、ssc-miR-1307、ssc-miR-23a等miRNAs的靶基因参与了免疫应答、细胞凋亡、细胞自噬、应激反应、细胞分化等重要的生物进程。结合KEGG通路分析,我们发现差异表达的miRNAs对MAPK通路、T细胞受体途径、Toll样受体途径、凋亡和自噬的调控起着重要的作用。该研究为miRNA在猪场实际生产中的应用提供了参考依据和新的思路,并为进一步进行仔猪免疫应激调控的分子机理研究,揭示猪miRNA介导的调控功能奠定了基础。更多还原

【Abstract】 With the rapid development of modern pig industry, piglet early weaning has become the key technology of intensive pig production at home and abroad. Weaning piglets, however, because of the immature immune system, susceptible to raise a variety of pathogens in the environment, causing immune stress, used in growth and skeletal muscle protein deposition of nutrients to maintain the body’s immune response, lead to the metabolic behavior changes, resulting in slow growth and development and production efficiency to reduce, to animal husbandry bring serious economic losses, directly affect the pig production level and economic benefits. Immunological stress seriously affect the growth of the thymus gland, thymus as central immune organ play an important role in immune regulation and control, the present study mainly concentrated in protein-coding genes, the miRNA mediated transcription level for immunological stress after molecular mechanism provides a new perspective. This study through the establishment of immunological stress model of weaning piglets, from screening related difference expression of micrornas in the thymus, and carries on the appraisal and analysis of biological information, the research result is helpful to further clarify the molecular mechanism to weaning piglet immune stress, as well as using the miRNA and its target genes information providing a new method to reduce the weaning piglet immune stress. In this study the main results were as follows:1.6Yorkshire weaning piglets weaned (21days) according to the tire same, birth weight, were randomly divided into experimental group and the control group, each group3pigs. Patients (including100g/kg BW LPS injection, control group the same amount of saline injection,3h, after weaning piglets thymus and other various organizations. Detection experimental group and control group in piglets serum interleukin-2, interleukin-6, prostaglandin and growth hormone found that the content of plasma inflammatory cytokines in the experimental group than control group in immunological stress secretion to increase, and growth hormone secretion was significantly decreased. That means after LPS stimulation, patients weaning piglets produced acute immune stress, inflammation reaction. 2. In the experimental group and control group in piglets thymus total RNA was extracted after we using Solexa sequencing technology to experimental group and control group of micrornas library sequencing, the results obtained up to14.5and14.5m respectively reading short sequence.126new miRNA is obtained by depth of Solexa sequencing control group, experimental group received149new micrornas. Solexa sequencing of the control group and experimental group a total of15expressed significant difference of micrornas, were tested, using qPCR results consistent with the Solexa sequencing results. That may play an important role in the immune stress expression profile build two micrornas in organization.3. After verification of miRNA target genes prediction and analysis found that WEGO function ssc-miR-129-a, ssc-miR-1271, ssc-miR-1307and ssc-miR-23-a target genes of miRNAs are involved in the immune response, cell apoptosis, cell autophagy, stress reaction, cell differentiation, and other important biological processes. KEGG pathway analysis, we found that differentially expressed miRNAs, T cell receptor pathway of MAPK pathway, toll-like receptor pathway, apoptosis and the regulation of autophagy plays an important role.The study for micrornas in pig farms in the practical application provides reference basis and new ideas, and laid a foundation on molecular mechanism of stress and regulating function of pig micrornas mediated for further piglet immune regulation research.更多还原