轮状病毒感染与inv基因沉默共同作用致胆道闭锁的机理研究

Rotavirus atresia (BA) is the most common progressive obstructive cholangiopathy in neonates with extremely poor prognosis. Recent researches show that "perinatal viral infection-immune abnormality" and "malformation of bile ductal plate" may both contribute in the development of BA, however none of the above can independently explain the pathogenesis of BA. Based on the above theories and our preliminary data, this project aims to establish a murine BA model based on perinatal rotavirus infection in pregnant mice and inv gene silencing in fetal mice, to investigate the relationship between the changes of inversin/Dvl1/beta-catenin and the severity of biliary injury/rotavirus infection, and to study the roles of inversin in regulating integrins and in mediating immune responses on bile ducts. Furthermore, this project aims to establish an in vitro cholangiocyte injury model based on inv gene silencing and rotavirus infection, to reveal the roles of inversin/Dvl1/beta-catenin/integrin signaling pathways in rotavirus-induced primary bile duct injury and immunocyte-mediated secondary cholangiopathy, and to understand the causal relationship within inv gene down-regulation-->beta-catenin decrease-->integrin increase-->rotavirus infection aggravation-->increased inflammatory responses-->development of BA. Therefore, our project is to integrate and improve the theories of "perinatal viral infection-immune abnormality" and "malformation of bile ductal plate" and to shed light on a new research direction in revealing the pathogenesis of BA.

胆道闭锁(BA)是新生儿最常见的进行性梗阻性胆管病变，预后极差。研究显示，“围产期病毒感染-免疫异常”及“胆管发育异常”均可致小鼠发生BA，但均不能独立阐明BA的发病机制。本项目基于上述理论及预实验结果，在孕鼠感染轮状病毒(RV)的同时，沉默胎鼠inv基因致inversin下调，研究inversin/Dvl1/β-catenin与胆管损伤/病毒感染的关系，探讨inversin对整合素的影响及与胆管炎症的关系。继而建立RV感染及inversin下调所致的胆管细胞损伤模型，阐明inversin/Dvl1/β-catenin/整合素信号通路在RV所致的原发损伤及免疫细胞介导的继发损伤中的作用，从而明确inv基因下调→β-catenin下调→整合素上调→RV感染加重→炎症加剧→BA的因果关系，为完善整合“围生期感染－免疫异常”和“胆管发育异常”学说，明确BA的发病机理开辟新的研究方向。

胆道闭锁的发病机理目前主要有两种学说即围产期病毒感染－免疫异常学说及胚胎期胆管发育畸形学说。前者认为轮状病毒感染后，CD8+T细胞、NK细胞及巨噬细胞等通过直接杀伤或免疫因子介导造成肝外胆管的持续性损害，从而导致胆道闭锁。但轮状病毒感染导致的胆道闭锁，肝小叶间胆管并无缺失，亦无小胆管增生，此病理变化与人胆道闭锁并不完全相符。后者认为inv基因的隐性缺失能导致肝外胆道连续性中断、小叶间胆管缺失及小胆管增生、肝内胆汁淤积，但却无肝外胆管炎症或胆管上皮坏死，这与人胆道闭锁病理变化又有所不同。因此我们使用siRNA技术下调胎鼠inversin蛋白的表达，并同时接种轮状病毒，拟整合胆道闭锁“围生期感染－免疫反应异常”和“胆管发育异常”学说。正式实验中构建上述模型更加符合人胆道闭锁的小鼠模型，通过观察胎儿期inv基因沉默小鼠胆管在出生后感染轮状病毒形成的胆道闭锁模型，及胆管上皮细胞的inv基因被沉默后感染轮状病毒的细胞模型，我们发现：（1）我们的inv-轮状病毒模型出现的肝脏损伤较传统模型重，其出现肝细胞水肿、变性的程度较传统模型鼠重，汇管区炎症细胞浸润亦较重，发生上述改变的时间节点较传统模型鼠早。（2）早期小鼠肝脏组织中的轮状病毒的滴度及蛋白水平较传统模型高，证实了inv基因敲除时，轮状病毒能更早更快的进行对肝脏及胆管细胞进行损伤，同时增高的胆红素水平及黄疸水平也表明梗阻症状更重。（3）同时，此模型小鼠胆管细胞的炎症及凋亡水平均比传统模型鼠重。（4）上述作用是通过inversin/Dvl1/β-catenin/整合素α2β1信号通路发挥作用的。.本模型的建立，是印证了我们的猜想，即胆管发育基因的异常与围生期病毒感染的共同作用可能导致胆道闭锁的发生，而且此疾病模型同时具备肝外胆管发育畸形、狭窄及胆管系统炎症的表现，这也为我们提供了更加合适的早期筛查及治疗靶点，为胆道闭锁的临床预防与诊治提供了更广阔的思路，为明确胆道闭锁发生的病理机制开辟新的研究方向。