条件性基因敲除与轮状病毒感染共同作用致小鼠胆道闭锁模型的建立与优化

Biliary atresia (BA) is a progressive obstructive cholangiopathy in intrahepatic and extrahepatic bile ducts with poor prognosis. So far, no individual animal model can mimic the pathological changes of BA perfectly, including bile duct ligation, sclerosing agent injection, gene knockout or rotavirus infection. Based on our previous etiological studies, we aim to establish an innovative murine model of BA by combining key gene knockout and rotavirus infection. Firstly, candidate genes associated with biliary development will be knocked down in.biliary epithelia followed by rotavirus infection, in which 2 to 3 genes will be identified by evaluating the degree of biliary injury and its following immunoreaction. Secondly, we will create or obtain these conditional gene knockout mice, and perform intra-peritoneal injection of various doses of rotavirus in newborn knockout mice and their controls. The growth, hepatic pathological changes and activation of immunocytes will be observed. Finally, we will then compare the hepatic pathological changes between the murine BA and human BA, and we hope to identify the best combination of proper dosage of rotavirus and the most vulnerable candidate genes. After the reliability and repeatability are proven, we hope to acquire a murine BA model which has consistent pathological changes with human BA, which would lay the foundation for future therapies for BA associated hepatobiliary injury.

胆道闭锁是一种预后较差的肝内外胆管闭塞性病变，但发病机制不明。以前采用的胆管结扎或药物注射、基因敲除、轮状病毒感染等方法诱导的动物模型均无法完整模拟人类胆道闭锁的病理改变过程。本研究拟在前期对胆道闭锁病因学研究的基础上，创新性采用基因敲除与轮状病毒感染共同作用的方式诱导小鼠胆道闭锁模型：首先在胆管上皮细胞中敲低与胆管发育相关的基因，并用轮状病毒感染上述细胞，通过评估胆管上皮细胞损伤程度及引发的免疫反应等找到2-3个候选基因；制作条件性基因敲除鼠，对这些基因敲除及对照组新生鼠腹腔注射不同剂量的轮状病毒，观察小鼠生长情况、肝脏病理改变及免疫细胞活化情况，并与人类胆道闭锁的肝脏病理改变相比对，找到与人类胆道闭锁病理改变相一致的轮状病毒感染剂量与基因敲除最佳组合；并研究该动物模型的可靠性和可重复性，从而获得与人类胆道闭锁病理改变相一致的小鼠模型，为开发出减轻胆道闭锁肝脏病损的治疗方法奠定基础。

胆道闭锁是发生于新生儿肝内外胆管炎症性闭塞性疾病，发病机制尚不清楚。建立良好的动物模型是研究胆道闭锁发病机制和治疗策略的重要手段，但现有的胆管结扎、基因敲除、轮状病毒感染等方法还不能完整模拟人类胆道闭锁的病理改变过程。本研究采用基因敲除小鼠模型结合轮状病毒感染的方式建立小鼠胆道闭锁模型，通过此模型研究遗传因素和环境因素相互作用导致胆道闭锁发生发展的机制。首先结合GWAS研究分析，发现Add3基因可能在BA发病中起重要作用。用轮状病毒感染Add3基因敲低的胆管上皮细胞，并与NK细胞共培养，发现 TNF-α和穿孔素的表达较低；进而构建Add3基因沉默的小鼠，并用轮状病毒感染，发现Add3基因沉默的小鼠肝脏组织中TNF-α、穿孔素表达水平很低，并且肝纤维化程度更严重；采用CRISPR/Cas9系统构建Add3基因敲除鼠，从蛋白水平和基因水平验证基因敲除效率；对基因敲除及野生型新生鼠腹腔注射不同剂量的轮状病毒，诱导胆道闭锁的发生，观察小鼠生长情况、胆道闭锁发生率、肝脏病理改变及免疫细胞活化情况，结果表明Add3基因敲除小鼠感染轮状病毒时胆道闭锁发生率明显高于野生型小鼠造模组，肝脏组织的病理分析显示早期主要发生门管区炎症细胞浸润，胆管上皮细胞受损，后期肝纤维化程度更为严重；接着重复该动物模型，研究其可靠性和可重复性，获得可以发生肝纤维化的小鼠。透射电镜和免疫荧光染色等发现Add3基因敲除小鼠与野生型小鼠胆管上皮细胞骨架和细胞间连接差异，Add3基因敲除鼠胆管上皮细胞间紧密连接更为疏松，门管区聚集的轮状病毒更多，并且免疫功能存在异常。我们以此模型为基础继续研究胆道闭锁的发病机制，为开发出减轻胆道闭锁肝脏病损的治疗方法奠定基础。