Foxp3+Treg新型效应分子FGL2作为泡球蚴病预后监测分子标记物的免疫学机制研究

Alveolar echinococcosis (AE) is a rare, but life-threatening zoonotic helminthic disease resulting from the intra-hepatic proliferation of the larval stage of the cestode, Echinococcus (E.) multilocularis. Ultrasound is the current method for regular imaging follow-up of AE, and is usually complemented by the more expensive computerized tomography (CT) at longer intervals. If available, PET-CT and serology byEm18-ELISA can provide some information on the activity status of E. multilocularis in response to treatment of inoperable lesions. A new cheap and accurate biomarker might nevertheless yield a better understanding of the immune response after E. multilocularis infection, and thus also a more direct information on the metacestode activity status. In a preliminary, explorative study with a limited number of AE patients, we got indication that both Foxp3+Treg and its new effector molecule Fibrinogen Like Protein2 (FGL2) were highly expressed in active AE patients, and became lower after surgery and in the inactive group. The aim of this project is to further explore whether FGL2 can be a good marker to follow up AE patients and how it works on the immune system, thus we intend to follow up 30 cases of AE patients (divided into an active and an inactive group) to tackle a correlation between FGL2 expression (assessed by serum-FGL2-ELISA) and the E. multilocularis activity (PET-CT scan). To study the functional role of FGL2, we will use a corresponding knock-out mouse model. Mice deficient in FGL2-expression will be infected with E.multilocularis, using wild type animals as a control group. E. multilocularis activity will be determined by micro PET-CT and RT-PCR with the 14-3-3-gene, FGL2-expression and IgG-production will be tested by serum ELISA; Foxp3+Treg, Th17, Th1 and Th2 cell typing will be carried out by Flowcytometry and qRT-PCR; inflammatory infiltration and pathological injury by HE staining. We anticipate that our findings will contribute to the understanding of how the host immune response contributes to the control of the disease, and how immune parameters can be used to assess prognostically the course of disease, and finally if immunological tools could also be used to improve the treatment of AE.

泡型包虫病（AE）是"类肝癌样"致死性寄生虫病，目前缺乏准确的预后监测分子标记物。我们前期研究发现Foxp3+Treg及其新型效应分子人纤维介素（FGL2）在活性期AE病人高表达，术后及非活性期表达较低。本项目拟随访30例AE病人，FGL2表达与PET-CT影像学联合检查，确定AE病人血清中FGL2表达量变化对AE病人预后监测的临床指导意义；利用体外抑制实验研究FGL2对T细胞增殖和抑制的影响及与Foxp3+Treg细胞之间的关系；通过泡球蚴感染FGL2基因敲除小鼠模型，利用小动物PET-CT及14-3-3基因评价Em活性，运用流式细胞术等手段研究不同感染时期FGL2 对 T细胞免疫（Foxp3+Treg、Th17、Th1、Th2）、特异性体液免疫（IgG1、IgG4）、炎症浸润及泡球蚴活性影响，揭示FGL2作为评价泡球蚴活性及预后监测分子标记物的免疫学机制，为包虫病预后监测提供科学依据。

泡型包虫病（AE）是“类肝癌样”致死性寄生虫病，其生长速度与寄生虫宿主免疫介导过程直接相关。我们前期研究发现，CD4 + CD25 + Treg新型效应分子纤维蛋白原样蛋白2（FGL2）在泡球蚴感染小鼠肝脏中过表达，但机理不明。我们使用FGL2基因敲除小鼠（fgl2-/-）研究发现，与泡球蚴感染野生型小鼠比较，泡球蚴感染FGL2基因敲除小鼠：（1）泡球蚴载量及增殖能力显著降低，（2）对ConA反应后T细胞增殖反应增加，（3）Treg数量及抑制功能均降低，（4）Th1型免疫反应占主导，DC细胞成熟增加。对肝脏病灶周围及远端各细胞因子及趋化因子的研究表明，在泡球蚴感染早期主要以IL-12a，IFN-γ和IL-4的混合Th1/Th2免疫应答。感染中晚期呈现出以IL-5，IL-10和TGF-β为主导的免疫耐受状态。IL-17在肝脏中持续性表达，主要分布在病灶周围炎症浸润带。对病灶周围细胞因子和趋化因子的全面研究将有助于筛选新的可能的免疫治疗靶标。对AE病人血清中FGL2表达水平的研究发现，AE患者的FGL2血清水平显著高于健康对照组，而治愈后FGL2水平显著降低。在AE患者中，FGL2的血清水平与Em18显著相关，提示可以作为AE患者长期随访的标志物之一。