细粒棘球绦虫原头蚴定向发育的调控机制研究

Echinococcus granulosus, as a member of the parasitic platyhelminth, is the causative agent of one of the most prevalent and fatal zoonotic disease throughout the world. It is highly prevalence rate in cattle population in western and northwestern China. Although the infection cycle of E. granulosus has been well described, the mechanism underlying the main developmental events remains largely unknown. By the whole genomics and transcriptomics sequencing, our previous study firstly revealed that the numerous genes were differentially expressed in four different life-cycle stages, and some of them exhibited stage-specific transcripts. However, the developmental processes of E. granulosus are more complex than other platyhelminth parasite. Protoscleces are capable of developing into adult worms in the gut of the definitive host after bile stimulated. They can also develop into secondary hydatid cysts if rupture and content leakage from the primary fertile cyst in the intermediate host. To explain this alternative development, we plan to used Solexa high-throughout sequencing technology to sequence the mRNA and miRNA transcriptom from the protoscleces with or without bile stimulation. Both groups samples are collected at 0 hour, 12hour, 24 hour, 7day and 14 day. We also propose to utilized a sensitive realtime-PCR for quantitative analysis of the expression patterns of the crucial genes and miRNAs across two treatment processes. Furthermore, by the informatics tools, we prepare to investigate the joint profiling of miRNA and their targets mRNA expression. This work sets the scene for the study of gene regulation in E. granulosus and provides a new approach to study the molecules involved in protoscleces development. It may also help us to find new strategies for the control of cystic hydatid disease.

包虫病是由棘球绦虫感染引起的人畜共患性寄生虫病，呈世界性分布。我国西部12个省（区）为高发区，流行区人口达6600万。为了攻克该病，本课题组在国家973项目支持下完成了细粒棘球绦虫的基因组精细图构建，分析了四个不同发育时期基因和miRNA的表达，从基因组水平上描绘了棘球绦虫阶段发育的遗传特征。由于棘球绦虫具有在不同宿主体内独特的双向发育特性，即原头蚴在中间宿主-羊或人体内发育成包囊，而在终宿主-狗胆汁刺激下发育成成虫。目前其定向发育调控机制还不清楚。因此，本研究拟通过检测原头蚴在加胆盐和不加胆盐两种体外培养条件下和多个时间节点的基因和miRNA表达的动态变化，寻找对棘球绦虫发育分化具有调控作用的关键基因和miRNA；同时，通过生物信息学分析，探寻miRNA所调控的关键靶基因，拟揭示棘球绦虫发育分化的调控机制，及其与宿主的相互作用关系。本课题的开展将为今后包虫病和其它寄生虫病的防治奠定基础。

包虫病是严重危害社会和人民健康的人畜共患病，本研究我们在前期基础上从基因组、转录组、转录后修饰（miRNA）三个水平对包虫病致病寄生虫之一-细粒棘球绦虫的发育机制进行研究，发现以下成果：①通过对细粒棘球绦虫的全基因组检测，建立了151.6Mb全基因组数据库，并成功预测11,325个功能基因。发现包虫体内核受体家族基因与胆汁酸盐代谢有关，成功鉴定包虫的钙离子通道蛋白（EgCaB），并发现其与包虫病治疗药物吡喹酮的敏感性有关；②通过检测细粒棘球绦虫三个不同的发育时期，新鉴定94个包虫的miRNA，发现65个成熟的miRNA在各时期间表达有差异，进化分析发现细粒棘球绦虫所丢失的miRNA基因家族与某些进化消失的特定组织器官有关；③通过体外培养，检测原头蚴在加胆汁和不加胆汁两种条件下0小时、24小时、7天和14天4个时间节点的基因表达谱，发现3,219个基因存在可变剪切，该比例明显高于其他非寄生扁平动物门生物，从而提示包虫可以通过增加自身基因的可变剪切使其更适应寄生生活，GO功能富集分析我们发现细粒棘球绦虫成虫发育的差异表达基因主要集中在神经系统发育和碳氢化合物代谢，而成囊的差异表达基因则主要与细胞外的物质代谢有关。④同一模型，我们还检测不同时间节点miRNA的表达变化，发现9个新的细粒棘球绦虫miRNA，其中3个miRNA属于已知的miRNA超家族，比较成囊和成虫两个发育方式的miRNA表达，分别发现31个和27个miRNA与两种发育方式相关，比较他们的表达模式发现12个miRNA在成囊和成虫两种发育方式中有差异，发现miR-71在包虫成囊发育明显升高，可能与调控宿主的免疫有关，发现let-7在成虫发育初期明显升高，可能与原头蚴的定向发育有关。以上成果无疑帮助我们对包虫病的发病机制有了更加深入的认识，也为今后包虫病的基因治疗奠定基础。