哈萨克绵羊抗包虫病个体小肠差异表达基因、miRNA筛选及抗性验证

Sheep hydatidosis (echinococcosis) is recognized as one of the word major zoonoses, which known as "second cancer", and is a serious threat to the health of husbandry and herdsmen in Xinjiang. Preliminary studies have shown that, in Kazakh sheep population, the effect of MHC-DRB1 MvaIbc-SacIIab-Hin1Iab haplotype on the resistance of hydatidosis was highly significant, but this relationship still needs a further validation. Resistance or susceptibility of sheep MHC genotype is related to the strength and type of the lymphocyte immune response, when the MHC antigen expressed by MHC in the small intestinal mucosa combine with oncosphere antigen and then both of them are presented to lymphocyte，as well as genes and miRNA related to MHC regulation in the small intestinal mucosa. In this study, the small intestine tissues represent the stages when oncosphere invasion the small intestinal mucosa from the resistance or susceptibility Kazakh sheep are used as experimental materials，presenting a global survey of the differentially expressed genes and miRNA with DGE and miRNA Solexa depth sequencing, and determine the role of miRNA regulation of MHC gene. In order to explore the molecular mechanism of small intestine against the oncosphere infection in resistance sheep, and further verify the resistance of the MHC resistance genotype for hydatidosis in Kazakh sheep, and provide a theoretical basis for the use of marker-assisted developing new sheep breed which is resistant to hydatidosis.

绵羊细粒棘球蚴病（包虫病）是一种人畜共患病，有"次癌"之称，严重威胁着新疆畜牧业生产和农牧民的身体健康。前期研究表明，新疆哈萨克绵羊MHC-DRB1的MvaIbc-SacIIab-Hin1Iab基因单倍型与包虫病抗性极显著关联，这种相关性还需要进一步验证。绵羊MHC基因型的抗性或易感性首先与小肠粘膜中MHC基因表达的MHC抗原与六钩蚴抗原结合后递呈给淋巴细胞产生免疫反应的强弱、类型有关，同时也和小肠粘膜中与MHC基因调控相关的其他基因以及miRNA密切关联。本研究拟利用数字基因表达谱和Small RNA测序的方法，筛选出包虫病抗性与非抗性绵羊在六钩蚴进入小肠粘膜时期差异表达的基因和miRNA，并确定调控MHC基因的miRNA的作用，从而探讨抗包虫病绵羊小肠抵抗六钩蚴感染的分子作用机制，并进一步验证哈萨克绵羊MHC包虫病抗性基因型的抗性，为利用标记辅助选择选育绵羊抗病新品种提供理论依据。

绵羊细粒棘球蚴病（包虫病）是一种人畜共患病，有“次癌”之称，严重威胁着新疆畜牧业生产和农牧民的身体健康。前期研究表明，新疆哈萨克绵羊MHC-DRB1的MvaI bc-SacII ab-Hin1I ab基因单倍型与包虫病抗性极显著关联，但具体机制尚不清楚。为了研究不同MHC基因型哈萨克绵羊产生包虫病抗性差异的具体机制，本研究主要进行了以下工作：①.利用RFLP-CPR技术筛选获得了对包虫病呈抗性（实验组，A组）和非抗性（对照组B组和空白对照组C组）绵羊，建立包虫病人工感染动物模型；②.利用转录组测序和Small RNA测序的方法，筛选出了包虫病抗性与非抗性绵羊在六钩蚴进入小肠粘膜时期差异表达的基因和miRNA，并对二者进行联立分析，选择其中与免疫反应相关的差异miRNA及其调控的差异表达基因，探讨它们与个体包虫病的相关性。生物信息学分析获得了大量差异表达基因，主要富集在Natural killer cell mediated cytotoxicity、Metabolism of xenobiotics by cytochrome P450、和Intestinal immune network for IgA production等通路，而差异miRNA调控的差异表达基因则与Metabolic pathways、Cytokine-cytokine receptor interaction和HTLV-I infection等通路密切相关，此外，与B组相比，A组中PPA1、BDH1和KIR2DS1等基因高表达也值得关注；③.利用各组的小肠组织匀浆液检测免疫指标并分析其变化规律，结果表明抗病和非抗病个体表现出不同的免疫应答能力。A组绵羊（MHC包虫病抗性组）表现出以IgE、IgG， Th1细胞因子（IL-2、IFN-γ、TNF-α）为优势分泌水平，而B组（非抗性组）则表现出以Th2细胞因子（IL-4）为优势分泌水平；④.利用免疫组化技术检测了各组绵羊感染包虫病后小肠组织的IgA、 IgG和IgM抗体表达差异，结果发现抗性组绵羊肠道组织中三种抗体的分泌量比非抗性组更高。且感染时间的增加，抗性组分泌量呈现升高趋势，但非抗性组未见明显变化。本研究认为，A组与B组免疫反应相关的多种基因、抗体和细胞因子的表达差异是导致不同哈萨克绵羊个体呈现包虫病抗性的主要原因。