棘球蚴囊壁生发层及原头蚴的AQPs在棘球蚴液生成中的作用及机制研究

Hydatid cysts, the matacestodal larvae of Echinococcus granulosus (Eg), are the etiological agents of cyst echinococcosis, also known as cyst hydatidosis, a severe zoonotic parasitosis. The origin and formation mechanisms of hydatid fluid in the growth process of hydatid cyst remain a mystery. Recent studies have found that E. granulosus possess genes of enconding aquaporins (AQPs), however, neither the expression of AQPs on the germinal layer of hydatid cyst wall nor the relationship between AQPs and the formation of hydatid fluids have been reported yet..In the present study, we plan to detect the expression of AQPs genes on the germinal layer of hydatid cyst wall by real-time quantitative PCR to investigate the expression patterns of AQPs on the germinal layer. The genes of AQPs on the germinal layer will be cloned by PCR, and will be expressed heterologously in Xenopus oocytes to measure the transporting vitality of AQPs for determining whether or not the typical characteristics of AQPs could be found in the hydatid cyst wall. .Based on the fact that a secondary hydatid cyst can be resulted from a protoscolex, we will simulate the development process from protoscolex to hydatid cyst in vitro. The expressions of AQP genes of protoscolex will be silenced by RNA interfere (RNAi). The survival rate of protoscolex, the time of cyst-forming, and the volume of hydatid fluids will be investigated to explore the possible mechanisms of relation between AQPs and producing of hydatid fluids. The ultrastructures of the protoscolieces silenced by RNAi, and the ultrastructures of germinal layers of cyst wall of secondary hydatid cysts from the protoscolieces with silenced RNA, will be observed by electron microscopies for a better understanding of effects of AQPs on development of germinal layers of hydatid cyst wall. And other interfering factors of AQPs expressions would be investigated in order to understand the possible roles of AQPs in the formation of hydatid fluids. The results of this research project may be helpful to understand the development process of hydatid cyst further, and may provide new ideas for the prevention and treatment of cyst echinococcosis.

棘球蚴是棘球蚴病（囊型包虫病）的病原体。在棘球蚴生长过程中其囊液的来源及生成机制一直是个谜。近来研究发现细粒棘球绦虫有编码水通道蛋白（AQPs）的基因，但尚无AQPs在棘球蚴生发层的表达及其与囊液形成关系的研究。.本研究拟通过实时荧光定量PCR方法检测AQPs基因在棘球蚴囊壁生发层的表达情况，阐明AQPs在生发层的表达模式。通过PCR克隆棘球蚴囊壁生发层AQPs基因，并在非洲爪蟾卵母细胞中进行异源表达，测定AQPs转运水分的活力，确定是否表现出典型的水通道蛋白特征。根据原头蚴可发育为次生棘球蚴的特点，体外模拟原头蚴发育为棘球蚴的过程，运用RNA干扰技术沉默原头蚴AQPs基因的表达，然后观察经RNA干扰处理的原头蚴存活率﹑原头蚴发育为棘球蚴的成囊时间及囊液量、囊壁生发层超微结构。以多种干预因素研究AQPs在棘球蚴液生成中的机制，阐明AQPs在棘球蚴液生成中的作用，为棘球蚴病防治提供新思路。

为了研究棘球蚴囊壁生发层及原头蚴的水通道蛋白（AQPs）在棘球蚴液形成中的作用及机制，本项目首先对EgAQPs基因进行生物信息学分析，发现12个EgAQPs蛋白可分为水通道蛋白及水-甘油通道蛋白两类，EgAQPs的与透水功能有关的NPA基序与经典AQP的两个保守NPA基序相比有缺失或改变现象。构建了原头蚴体外培养形成棘球蚴及小鼠接种原头蚴形成棘球蚴模型。用RT-qPCR方法检测12个EgAQPs基因在体内外不同生长时期棘球蚴的转录表达水平，结果显示各EgAQPs基因表达量均很低。以RNA-seq技术对次生棘球蚴进行转录组测序、分析EgAQPs表达量，发现除AQP3、AQP4和AQP9表达量稍高外，其余基因几乎没有表达，此结果证实了RT-qPCR检测结果。应用RT-qPCR检测体外培养的次生棘球蚴AQPs基因在不同渗透压处理下的相对表达量，发现渗透压对EgAQPs基因转录水平变化影响不大。以原头蚴cDNA为模板克隆12个EgAQPs基因并与质粒pCS107连接构建重组质粒，导入非洲爪蟾卵母细胞中进行EgAQPs基因透水功能验证，结果显示各基因均未表现出透水功能，且Western blot检测未见EgAQPs在爪蟾卵母细胞膜上表达，表达蛋白有被剪切现象。运用生物信息学软件分析发现EgAQPs基因密码子偏好性普遍较弱，提示酵母表达系统可能优于爪蟾卵母细胞。根据上述转录组分析及密码子优化分析结果，选择表达量高的EgAQP9-1以毕赤酵母异源表达，Western Blot 检测该基因在酵母表达系统的表达状况亦发现表达蛋白有被剪切现象。以自制的多克隆抗体检测发现EgAQP9可见于棘球蚴生发细胞的胞质、胞膜及原头蚴表面。综上所述，本研究发现在原头蚴囊化形成次生棘球蚴过程中EgAQPs基因可能是以低转录方式或者分子结构“失能”方式阻止水分子流出囊外，导致棘球蚴液逐渐积累。