miR-335通过靶向沉默NLRP3抑制华支睾吸虫感染致肝纤维化的研究

Clonorchiasis is one of the major parasitic diseases which significantly endangers people’s health. It mainly leads to hepatobiliary system disease, even liver fibrosis and cholangiocarcinoma. But the role of endogenous regulation mechanism in liver damage caused by Clonorchis sinensis is unclear. Our previous study found that Clonorchis sinensis infection caused hepatocyte apoptosis, hepatic iron deposition and miRNA abnormal expression. However, its mechanism had not been elucidated. We had found that miR-335 is significantly reduced by Clonorchis sinensis infection. In addition, miR-335 could inhibit the fibrosis regular molecular NLRP3. Therefore, we assume that miR-335 through the NLRP3/caspase-1 signaling pathways regulating liver fibrosis with Clonorchis sinensis infection. This project intends to apply the transgenic animals, Lentiviral vector system and coculture cell model, using Gene Chip, Western blot, Real-time PCR, Luciferase reporter gene technology, to confirm that the role of miR-335 in the process of liver fibrosis by Clonorchis sinensis infection. In addition, we would further study the mechanism of miR-335 in regulating the NLRP3/caspase-1 signal transduction pathway during the excretory secretory antigen of Clonorchis sinensis activated hepatic stellate cells. Through the above study, we expect to reveal that the mechanism of miR-335 targeted silence NLRP3 inhibit the liver fibrosis with Clonorchis sinensis infection, providing the new strategy for prevention and treatment of liver fibrosis caused by Clonorchis sinensis.

华支睾吸虫病是严重危害人民健康的重大寄生虫病，主要表现为肝胆系统病变，甚至发生肝纤维化、肝胆管癌。但宿主的内源性调控机制在该虫感染致肝损伤中的作用尚未阐明。课题组在已证实该虫感染致肝细胞凋亡、肝铁沉积和肝脏miRNA表达异常的基础上，进一步研究发现：1）miR-335在华支睾吸虫感染小鼠的肝纤维化组织中显著降低；2）miR-335重组慢病毒注射小鼠后肝纤维化程度减轻；3）miR-335对纤维化调控分子NLRP3有抑制作用。因此，我们假设miR-335通过NLRP3/caspase-1信号通路调节华支睾吸虫感染致肝纤维化。本项目拟应用转基因动物、慢病毒载体系统和共培养细胞模型，采用基因芯片、Western blot、Real-time PCR和荧光素酶报告基因等技术阐明miR-335在华支睾吸虫感染致肝纤维化过程中的作用与确切分子机制，为防治华支睾吸虫感染致纤维化研究提供新思路和可能靶点。

华支睾吸虫病是危害人类健康的重要食源性寄生虫疾病之一，且感染率呈上升趋势，黑龙江省是该病的高发区。长期慢性感染华支睾吸虫可导致宿主发生肝纤维化和肝胆管癌，患者预后较差。国际癌症研究署（IARC）于2009年将华支睾吸虫列为一类致癌病原生物，该虫成为11种一类致癌病原生物之一。因此本项目从宿主内源性调控机制的角度研究华支睾吸虫致宿主肝胆损伤的机制，研究结果将为该病的防治提供新思路和新靶点。本研究的主要内容包括：收集正常人、华支睾吸虫病患者尸检肝组织标本和华支睾吸虫病患者血清标本；制备华支睾吸虫感染模型，华支睾吸虫感染诱导肝纤维化模型；检测不同阶段华支睾吸虫感染宿主的肝胆损伤情况（肝纤维化和肝细胞凋亡）；检测华支睾吸虫感染后宿主肝组织的铁代谢变化，并分析肝铁沉积与宿主肝胆损伤、肝细胞凋亡和肝纤维化的关系；利用基因芯片检测华支睾吸虫感染后大鼠肝脏组织microRNA的表达变化情况，筛查差异显著、且具有研究意义的microRNA（miR-335）；检测华支睾吸虫感染大鼠肝组织NLRP3通路相关因子的基因和蛋白表达水平，阐明NLRP3信号通路在华支睾吸虫感染致宿主肝纤维化中的作用；结合miR-335在华支睾吸虫感染宿主过程中的变化情况，进一步阐明miR-335通过靶向沉默NLRP3抑制华支睾吸虫感染致肝纤维化中的作用，从而从宿主内源性调控机制的角度深入研究华支睾吸虫感染致宿主肝胆损伤的机制。