宿主信号分子BMP2通过细粒棘球蚴BMP/Smad信号通路调控虫体生长发育及其致病性的研究

Echinococcosis is a serious zoonoses in northwest of China and more than 95% cases are cytic echinococcosis (CE), which caused by Echinococcus granulosus (E. granulosus) infection. Xinjiang is a high risk epidemic region. Study on the effect of host signal molecule BMP2 on growth, development and pathogenicity of E. granulosus is rarely reported. In our previous study, we found that the high expression of BMP2 molecule was around lesions in CE patients with active stage; BMP2 promotes E. granulosus protoscolex development into cyst in vitro, and BMP/Smad signal pathway was in the presence of E. granulosus. It suggests that BMP2 may regulate the growth, development and high pathogenicity of E. granulosus through the BMP/Smad signaling pathway, but the mechanism is unknown. In this work, we plan to elucidate the effects of BMP2 on the encysted development of the primary cells of the germinal layer through the BMP/Smad signal pathway. Using human BMP2 stimulation and the inhibitor of BMPR I in E. granulosus cultivation system in vitro and in E. granulosus infection mice in vivo, we identify the molecular mechanism of E. granulosus growth, development and pathogenicity which was regulated by host BMP2, and verify the relationship between BMP2 and pathogenicity of E. granulosus by clinical specimens. Finally, we explore the feasibility and effectiveness for CE treatment by the use of BMP2 neutralizing antibody or inhibitor in the E. granulosus infected mice model in vivo. These studies have important scientific significance to reveal the mechanism of growth, development and pathogenicity of E. granulosus, and to develop new drugs and new therapeutic method.

包虫病是危害严重的人畜共患病，95%是细粒棘球蚴（Eg）感染所致囊型包虫病（CE），新疆是高发区。宿主信号分子BMP2调控Eg生长发育和致病性的研究未见报道。课题组前期研究发现活跃期CE病人病灶周围BMP2高表达，且BMP2促进体外Eg原头蚴向成囊方向发育；Eg自身存在BMP/Smad信号通路，提示BMP2可能通过BMP/Smad信号通路调控Eg生长发育及高致病性，但机制不明。本项目拟利用生发层原代细胞系，明确BMP2通过BMP/Smad信号通路对生发层原代细胞成囊发育的影响；其次Eg体外培养和体内接种动物方式，通过正向激活和反向干预方法，阐明BMP2调控Eg生长发育和致病性的分子机制，临床标本验证BMP2与Eg致病性的关系；最后使用BMP2中和抗体或抑制剂干预Eg感染的动物模型，探索用于CE治疗的可行性和有效性，对揭示Eg生长发育和致病机制、研制新型药物和开发新治疗方法具有重要科学意义。

包虫病是由棘球绦虫的幼虫寄生于人体所致的一种严重的人畜共患寄生虫疾病，病例中95%为细粒棘球蚴（Echinococcus granulosus, E. g）感染所致囊型包虫病（Cystis Echinococcosis, CE）。宿主信号分子BMP2调控Eg生长发育和致病性的研究未见报道。本研究首先成功建立细粒棘球蚴囊泡生发层原代细胞系，发现BMP2信号分子通过激活下游BMP/Smad信号通路，促进生发层原代细胞的增殖，以及多克隆集落的形成；利用该通路抑制剂干预诱导原代细胞凋亡。其次体外培养细粒棘球蚴原头蚴和囊泡，发现BMP2信号分子通过BMP/Smad信号通路促进原头蚴向成囊方向发育，有利于原头蚴细胞的增殖；该通路抑制剂干预实验表明LDN193189和DMH1对细粒棘球蚴均具有杀伤效果，其中LDN193189对原头蚴和囊泡两个阶段杀伤效果较好；借助高通量测序方法，研究发现宿主信号分子BMP2可能与虫体表面受体结合，依赖信号通路调控细粒棘球蚴细胞增殖、死亡和发育；其通路抑制剂DMH1能够干预蛋白质合成和代谢途径影响细粒棘球蚴生长发育过程。体内实验研究表明，细粒棘球蚴原头蚴经BMP2信号分子体外刺激后，对再感染小鼠腹腔虫体生长有一定促进作用；经抑制剂LDN193189干预后，对小鼠的感染率显著下降。接着成功建立细粒棘球蚴原头蚴经肝门静脉囊型包虫病小鼠模型，更好模拟临床患者表现，确定感染无剂量效应，以及小鼠体内原头蚴在肝脏向成囊方向发育的关键时间节点；通过腺病毒高表达BMP2能够限制小鼠肝脏病灶的生长。利用经肝门静脉和腹腔感染小鼠模型研究发现，LDN193189和DMH1抑制剂经灌胃途径有一定的治疗效果。最后利用CE患者肝脏手术标本，证实BMP2信号分子及其下游BMP/Smad通路激活与CE患者临床活跃性的关联性，参与宿主肝脏囊泡的生长发育。本项目初步揭示宿主信号分子BMP2通过BMP/Smad信号通路调控细粒棘球蚴生长发育和致病的分子机制，对研制新型疫苗和药物、探索囊型包虫病新的临床方法均具有一定的科学意义。