断奶仔猪免疫器官应激过程解析及SKP2基因作用机理研究

In process of protein degradation, SKP2 can regulate cell cycle proceeding due to recognizing and degrading cell cycle factors by constructing the component of SCFskp2 compound, it plays a considerable role in the development of organs and tissues. Early research results from applicant of this project show that not only polymorphic site of SKP2 gene has significant correlation with the index of immunity of piglets, but also SKP2 expression amounts in thymus, lymph node and spleen rank the top four in ten kinds of important organs. It indicates that SKP2 gene may have a considerable relationship with the development of porcine immune organs. Given that the effect of weaned piglets' stress challenge on swine production industry and its challenge mechanism remains to be elucidated. The model on weaned piglets' stress challenge will be constructed in this project, not only the essential correlation of SKP2 gene with tissue proliferation and apoptosis of weaned piglets' immune organs but also the role on SKP2 gene to tissue and cell differentiation will be investigated by biological histology, immunohistochemistry, electron microscope, molecular biology techniques and methods on the basis of tissue, cell, sub-cell and molecular level, moreover, the biological function of porcine SKP2 gene will be initially found out. The stress challenge process of weaned piglets' immune organs and action mechanism on porcine SKP2 gene will be compositely analyzed. The molecular basis of easy pathogenicity to weaned piglets in production practice will be ascertained, and it will provide scientific theory proof for revealing the pathogenesis to weaned piglets and its molecular theory for disease resistance.

在蛋白降解过程中，SKP2通过构成SCFskp2复合物的成份识别和降解细胞周期因子而调控细胞周期进程，对器官组织的发育具有重要作用。项目申请人的前期研究表明不仅存在SKP2基因的多态位点与仔猪的免疫指数相关联，而且胸腺、淋巴结和脾脏的SKP2表达量位列十种重要器官的前四位，提示SKP2基因与猪免疫器官的发生发育可能存在重要联系。考虑到断奶仔猪疾病应激对养猪业的影响及其应激机理尚未阐明，本项目拟建立断奶仔猪免疫应激模型，采用生物组织学、免疫组织化学、电镜及分子生物学技术和方法，从组织、细胞、亚细胞及分子水平研究SKP2基因与仔猪免疫器官组织增殖和细胞凋亡存在的本质关联及其SKP2基因对组织细胞分化的作用，初步探明猪SKP2基因的生物学功能，综合解析仔猪免疫器官的应激过程及SKP2基因的作用机理，探明生产实践中断奶仔猪易致病性的分子基础，为揭示断奶仔猪的发病机制和分子抗病机理提供科学理论依据。

本项目主要研究了LPS（脂多糖）诱导断奶仔猪主要免疫器官脾脏的宏观形态与微观结构的影响，及其组织发育与猪SKP2基因表达的关联，通过亚细胞定位检测了SKP2蛋白的表达位置，分析SKP2基因在细胞内的作用。运用超表达技术检测转染脾细胞中SKP2基因的表达及细胞凋亡情况，预测分析了SKP2蛋白的保守功能结构域。主要结果是：（1）在LPS诱导断奶仔猪的过程中，外周免疫器官脾脏和淋巴结具有形态增大、颜色加深、重量增加等外形变化，而其它器官变化不显著。脾小结数量明显增多、增大、PALS增厚、边缘区变薄，局部出现淤血现象。（2）SKP2表达水平升高，而P27Kip1表达水平减弱，脾脏淋巴细胞增殖分化，增强机体免疫；凋亡蛋白Caspase-3活性受到抑制，脾脏细胞凋亡进程受阻。（3）经过Lenti-skp2-EGFP慢病毒感染脾脏细胞的流式检测细胞凋亡，发现脾脏细胞凋亡率降低，表明SKP2基因的过表达抑制了细胞的凋亡，与活体仔猪应激模型具有一致的结果。亚细胞定位SKP2基因的荧光表达产物存在脾细胞核内，SKP2蛋白具有一个保守的F-box结构域，可能通过该结构域在细胞核内行使蛋白识别与降解的功能。.断奶仔猪免疫器官脾脏对LPS的诱导极为敏感，迅速表现出其免疫应变能力。在此过程中，SKP2抑制状态被释放从而呈高表达水平，脾脏出现增殖，致使其大体形态和显微结构均显著变化，表明SKP2基因在断奶仔猪的致病过程及免疫器官状态改变中具有调节分化的作用。