miR-133a和TGF-β介导的泡球蚴感染所致宿主肝损伤的分子机制及其病理学意义

Alveolar echinococcosis (AE) is characterized by the tumor-like growth of Echinococcus multilocularis (Em). Very little is known about the influence of helminth parasites on the hepatic injury mechanism. In our previous study, we found that TGF-β indicated an increased expression during the various stages of infection, whereas miR-133a indicated a decreased expression. The inverse correlation between TGF-β and miR-133a expressions, suggest that they could be involved in the progression of hepatic injury, of which mechanism, very little is known to date. The aim of this work or project is to identify miR-133a, and indicate how to regulate its target gene, which is involved in the TGF-β signaling pathway. This will be achieved, by co-culturing Em / hydatid fluid and hepatocyte (HC) / Hepatic stellate cell (HSC), and then reveal, how miR-133a and TGF-β together, regulate host liver cells (HC/HSC) differentiation and apoptosis. In E. multilocularis "model", mice will be sacrificed by injecting them with miR-133a intravenously. During the various stages of the infection process we would clarify the hepatic injury rule, by which miR-133a and TGF-β are mediated. We would then decide whether or not, exogenous miR-133a is a therapeutic target in liver injury. The verification of clinical samples will prove the feasibility and effectiveness of miR-133a in the diagnosis of AE as a new biomarker. These results will lay the foundation for studying the pathogenic mechanism of chronic infectious diseases, clinical intervention and further research work.

泡型包虫病（AE）是泡球蚴（Em）寄生肝脏所致的"类肝癌样"致死性寄生虫病，肝损伤分子调控尚未阐明。我们前期研究发现：Em感染后诱导宿主肝脏TGF-β水平持续高表达，但miR-133a表达水平却被持续抑制，提示miR-133a和TGF-β介导参与了Em感染所致宿主肝病理损伤过程，但机制不明。本项目拟通过体外Em/囊液-肝细胞/肝星状细胞共培养体系，明确miR-133a对TGF-β信号通路关键靶基因调控，揭示miR-133a和TGF-β介导的宿主细胞的分化与凋亡；基于小鼠体内感染和外源导入miR-133a干预实验，阐明Em感染后不同时期miR-133a和TGF-β介导的宿主肝损伤机制，确定外源miR-133a作为小分子肝损伤治疗靶标的效用；验证临床样本miR-133a作为新的泡型包虫病肝损伤诊断分子标志物的可行性和有效性，为研究其他慢性感染性疾病致病机制，发现新的临床干预和随访靶标奠定基础。

泡型包虫病（AE）是泡球蚴（Em）感染宿主肝脏所致的致死性寄生虫病，肝脏损伤的分子机制不明，本研究（1）建立了原代肝星状细胞和肝细胞的分离培养及鉴定体系，发现Em蛋白能够抑制原代肝星状细胞的活化，下调miR-133a的表达。外源导入miR-133a能够下调活化型LX-2细胞中α-SMA、Col1A1和Smad4表达，抑制TGF-β1对肝星状细胞的活化。（2）建立了不同剂量泡球蚴经肝门静脉感染小鼠动物模型。泡球蚴感染能促进小鼠肝星状细胞活化，抑制miR-133a在肝星状细胞中的表达。外源导入AAV8-miR-133a在泡球蚴感染早期会减轻泡球蚴感染所致肝脏纤维化程度，但却促进小鼠体内泡球蚴成囊率。TGF-β表达随泡球蚴感染时间延长而增多，纤维化程度加重，TGF-β/Smad信号通路参与了泡球蚴感染不同阶段小鼠肝脏纤维化的病理损伤过程。低剂量泡球蚴感染时，宿主免疫应答可清除其寄生。高剂量感染时则会诱导宿主形成免疫耐受形成慢性寄生，免疫类型T1型（IFN-γ和TNF-α）、T2型（IL-4、IL-5和IL-13）和Treg型（IL-10、TGF-β1和FGL-2）细胞亚群相关细胞因子的表达显著高于低剂量组，而T17型（IL-17A）细胞亚群相关细胞因子表达显著低于假手术组。（3）在泡球蚴感染小鼠肝脏组织中发现69个差异表达miRNA。（4）在AE患者肝脏中筛选出441个差异表达基因，发现43个上调基因与AE患者肝脏炎症微环境炎症和纤维化相关，其中成纤维相关细胞因子（FGF21、FGFBP1和PDGFD）和炎症相关细胞因子（IFN-γ, TGF-β1和IL-10）表达上调。AE患者肝脏组织TGF-β1与IL-10表达和肝纤维化指标（α-SMA、Col I和Col III）呈正相关性。原位杂交检测结果显示miR-133a在病灶近旁肝脏组织中低表达。miR-133a在AE患者、CE患者和肝硬化患者的外周血中的表达量均高于健康志愿者。AE患者外周血miR-133a表达与患者血清中活化型TGF-β1、肝纤维化指标（COLA III）和肝功能指标（ALP）呈正相关性。本研究结果显示外周血miR-133a表达水平可作为潜在的泡型包虫病肝损伤诊断分子标志物，外源导入miR-133a具有抗肝纤维化的潜在用途，TGF-β和miR-133a协同介导参与了泡球蚴感染所致宿主肝损伤分子机理。