胆盐趋化细粒棘球绦虫原头蚴向成虫分化的分子机制

Hydatid disease (HD,echinococcosis) is caused by a dog tapeworm Echinococcus granulosus (Eg) which is a near-cosmopolitan distribution. In western China, HD is hyper-endemic with the prevalence reaching to 2-12%. As heavy economic burden on the family and communities, HD has been listed in free treatment scheme by the Chinese government and a control program has been commenced. However, as complicated life-cycle, social factors heavily involved in the transmission and a large number of intermediate hosts, it has been difficult to control the disease. Dog vaccination is considered as an ideal control measure. Isolation of vaccine candidate genes is first priority for the success. We have identified that bile salts play a crucial role in adult differentiation and development from protoscolex (PSC).In the study, we will use our established in vitro cultivation parasite model to address the molecular mechanisms underlying the differentiation and development of PSC to adult worm by sequencing the transcriptome in different time points during early adult development. The up-regulated and specific genes in the early adult differentiation and development will allow us to identify the key molecules for vaccine development for dogs against adult worm infection. The special aims of the project include (1)to determine the key components in bile salts, which induce Eg PSC developing to adult worms; (2) to determine the correlation of the key bile salts with worm differentiation and development; (3) identify transcriptome of Eg during the differentiation and development; (4) to identify 5 key genes up-regulated or specifically expressed in PSC during early differentiation to adults using RNAi technique； and (5) to express 3-5 genes encoding tegument proteins during adult development, and produce antisera to identify their possibility as vaccine candidates by in vitro killing assay using the serum.

包虫（棘球蚴）病由细粒棘球绦虫（Eg）引起，全球流行。我国西部高发流行，感染人群可达12%。该病由于社会因素的涉入，大面积流行和庞大的宿主群，控制极为困难。若能对犬免疫接种，将加速控制进程。寻找疫苗的靶基因是其关键。我们已证明胆盐是Eg 成虫发育和分化必需的趋化因子。本题研究拟利用体外模型确定胆盐诱导Eg 原头蚴分化过程中转录基因的表达水平，探讨成虫形成机制，筛选成虫分化关键基因，为犬抗Eg 疫苗的研制提供候选基因。研究内容包括(1)确定胆盐决定原头蚴向成虫分化的有效成分;(2)确定不同胆盐浓度与成虫分化的关系;(3)通过转录组基因的测序确定Eg 原头蚴分化过程中调控基因的表达水平,确定特异基因，正相调控基因以及相关的信号通路; (4)用RNAi确定选择基因在成虫发育中的作用；（5）表达3-5个成虫发育相关高表达的表面抗原，制备抗血清，通过体外杀伤试验，确定其疫苗研制的可能性。

包虫病病原细粒棘球绦虫的原头蚴具有双向发育的能力。在犬的肠道发育为成虫，而进入其他内脏组织，则发育为包囊。我们建立了细粒棘球绦虫双相发育的体外培养平台，本课题主要任务是探讨成虫发育的机制，重点探索胆盐对细粒棘球绦虫发育的调控和诱导机制。我们按计划完成了以下内容：1）探明原头蚴的成熟状态是成虫发育的关键因素，只有成熟的原头蚴才能发育为成虫，胃蛋白酶的消化可以得到成熟一致的原头蚴；2）原头蚴的外翻是成虫发育最关键的一步，低盐溶液可使大部分原头蚴外翻；3）外翻原头蚴的维持是成虫持续发育的最基本因素，短期的无血清培养可以阻止外翻原头蚴的折回再内翻；4）胆盐是成虫发育激活和维持的基本要素，原头蚴的双向发育是基于胆汁浓度的模式，在浓度大于0.2%时，成虫的发育受阻，对虫体有伤害或杀伤作用；低于0.01%时，原头蚴基本向成囊方向发展。5）不同单体胆盐对虫体的发育有显著的影响，牛黄胆酸钠是调节原头蚴向成虫发育的主要盐分，25uM牛黄胆酸钠是成虫发育的事宜浓度。其它盐对成虫的发育有抑制或毒害作用；6）成虫早期发育受胆盐正向调控的基因有361个，高表达的基因包括水通道蛋白，EgM123，膜联蛋白，胆盐协同转运蛋白，钙调蛋白，精氨酸酶，和胆盐相关的核受体蛋白。7）表达了水通道蛋白，EgM123，膜联蛋白，胆盐协同转运蛋白，HSP 和胆盐相关核受体蛋白。8)证明抗EgM123血清对原头蚴和成虫有抑制和杀伤作用，而抗HSP血清无杀伤作用；9)胆盐相关核受体的siRNA干扰使原头蚴致死。