TGF-β/Smad信号通路在细粒棘球蚴中的生物学功能及其干预研究

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. Until now, there is not any report of TGF-β/Smad signaling pathway in E. granulosus. In our previous study, three key members of Smads, EgSmadA, EgSmadC and EgSmadD, were cloned from E. granulosus and high expressions were found in the samples of germinal layer and protoscolex, which isolated from active CE patients. However, very little is known on biological function and pathogenic mechanism of TGF-β/Smad signaling pathway in E. granulosus. In this work, we plan to clone the TGF-β receptor I (EgTβR I) and II (EgTβ II) from the E. granulosus and detect the expression and location by RT-PCR and immunohistochemistry. Using yeast two-hybrid, we plan to identify the biological function and interaction of the key members of EgTGF-β/Smad signaling pathway. Using human TGF-β stimulation and the inhibitor of TβR I and Smad3 in E. granulosus cultivation system in vitro and in E. granulosus infection mice in vivo, we would like to know the influence of EgTGF-β/Smad signaling pathway on survival and pathogenicity of E. granulosus. Using RT-PCR and immunohistochemistry , we plan to detect the key member's activation of EgTGF-/Smad in 200 samples of CE patients to make sure the relation between the EgTGF-β/Smad signaling pathway activation and the pathogenicity of E. granulosus. Using TβR I and Smad3 inhibitors, we would like to know the anti-parasite possibility and effect of EgTGF-β/Smad signaling pathway interference in E. granulosus infected mice, which could be a potential anti-parasite drug or vaccine target. This is the first time we introduce TGF-β/Smad signaling pathway into CE research field and its result will enrich the knowledge of parasite signaling pathway network, which influence the pathogenicity of E. granulosus.

包虫病是危害严重的人畜共患病，其中95%是由细粒棘球蚴（Eg）感染所致囊型包虫病（CE），新疆是高发区，Eg中TGF-β/Smad信号通路研究尚属空白。前期研究提示Eg存在EgSmadA、EgSmadC和EgSmadD基因，在活跃期CE病人包囊中高表达，但功能不详。本课题拟克隆EgTβRⅠ和EgTβRⅡ受体，定位并检测表达；酵母双杂交等实验鉴定EgTGF-β/Smad通路关键成员的生物学功能及交互作用；Eg体外培养和体内接种感染动物方式，通过正向激活和反向干预实验，确定EgTGF-β/Smad信号通路对细粒棘球蚴存活和致病性的影响；利用临床CE病人标本验证EgTGF-β/Smad通路与其致病性的关系；使用抑制剂干预Eg感染小鼠模型，明确其作为新的抗包虫药物的可行性和有效性。本课题第一次将TGF-β/Smad信号通路引入CE研究领域，将极大丰富信号网络通路在包虫病致病机制中所起的作用。

包虫病是危害严重的人畜共患病，其中95%患者是由细粒棘球蚴（E. g）感染所致囊型包虫病（CE）。TGF-β/Smad信号通路在E. g致病机制方面的研究尚属空白，本课题旨在探讨E. g中TGF-β/Smad信号通路关键成员的生物学功能及其与致病性的关系。首先成功克隆E. g中TGF-β/Smad信号通路关键受体成员EgTβRI和EgTβRII，免疫组织化学显示主要表达定位于原头蚴体被和囊泡生发层；酵母双杂交、pull-down实验证实EgTGF-β/Smad信号通路关键成员之间存在交互作用，且EgTβRII能够与宿主信号分子HsBMP2结合，体外rHsBMP2刺激下游成员EgSmadE发生磷酸化。其次利用E. g体外培养和体内接种感染动物方式，发现EgTGF-β/Smad信号通路抑制剂对细粒棘球蚴原头蚴有较好的杀伤效果，显著降低虫体在小鼠腹腔的感染率。最后利用临床CE患者标本，发现外周细胞因子TGF-β1水平显著升高，提示宿主信号分子TGF-β家族可能通过EgTGF-β/Smad通路参与E. g的致病性。本课题初步阐明EgTGF-β/Smad信号通路在E. g生长发育中的作用，通过抑制剂干预明显降低细粒棘球蚴对宿主的致病性，从而为EgTGF-β/Smad中关键成员为靶点的药物研发、疫苗研制以及临床治疗提供新的思路。针对宿主细胞因子TGF-β家族对E. g生长、发育和致病性的调控机制，以及抑制剂对CE的疗效，包括用药浓度、疗程和副作用等相关指标的检测，还需进一步研究。