慢性酒精摄入诱导的肠道细菌肝内移位通过NETs并激活TLR9促肝细胞癌发生的机制研究

Chronic alcohol consumption is one of the risk factors for hepatocellular carcinoma (HCC) , however, the reasons are still unsure. Intrahepatic infiltration of neutrophils and chronic inflammation which are associated with hepatocarcinogenesis are results of intrahepatic translocation of intestinal bacteria in alcoholism. The formation of neutrophil extracellular traps (NETs) is a way of the elimination of pathogens and death of neutrophils, and the damage of hepatocytes would be alleviated obviously when the NETs were inhibited. On the other side, Toll like receptor 9(TLR9), which was a newly confirmed receptor of NETs, play a critical role in some kinds of liver injury and many signs also implied relationships between TLR9 and carcinogenesis. Therefore, we put forward the hypothesis that the formation of NETs resulting from intrahepatic translocation of intestinal bacteria activate TLR9 is one of the reasons why the risk of HCC is increased in chronic alcohol consumption. In order to prove the hypothesis, we will carry out several researches as follow: To evaluate the affection of bacteria in alcoholic hepatocarcinogenesis, the intestinal bacteria will be eliminated in a chemical induced HCC and alcoholism mice model; Build a same animal model by a NETs transgene mice to further comfirm the role of NETs in alcoholism hepatocarcinogenesis. Lastly, to study the TLR9 signaling in causing alcoholic HCC, a TLR9 gene knockout mice will also used. The study will further clarify the reasons why the alcoholism persons are predisposed to HCC, and will provide a theoretical basis to protect alcoholic from HCC.

长期酗酒是肝细胞癌（HCC）发生危险因素。慢性酒精摄入时肠道细菌肝内移位继而可致肝内中性粒细胞浸润及慢性炎症，而后两者均同HCC发生相关。中性粒细胞胞外诱捕网（NETs）作为中性细胞清除病原体的形式之一，其形成显著促进肝损伤。Toll样受体9（TLR9）作为其受体，参与多种形式肝损伤并有迹象表明其与肿瘤发生相关。据此提出：肠道细菌肝内移位后通过NETs并激活TLR9，是慢性酒精摄入促HCC形成的原因之一。为证实假说，本研究将构建小鼠伴慢性酒精摄入的HCC化学诱发模型，同时清除肠道细菌，研究肠道菌对酒精性HCC发生的影响；建立NETs形成关键酶基因缺失小鼠酒精性HCC模型，研究此时NETs的作用；制造TLR9转基因小鼠酒精性HCC模型，研究TLR9在其中的作用。本研究的完成将进一步完善酒精促HCC发生原因，为HCC预防提供潜在靶点和理论依据。

肝细胞癌是我国最常见的中之一，WHO最新的年度报告指出，长期大量饮酒是HCC发生的独立危险因素之一。随着近年来乙肝疫苗的普及，未来乙肝相关HCC将逐渐减少，而酒精相关型HCC比例将大幅增加。因此迫切需要针对酒精相关HCC发病及其防治进行更为深入的研究。中性粒细胞的肝内浸润，是长期酒精摄入的重要特征性表现之一。研究显示慢性酒精摄入时肠道屏障受损，肠道细菌大量增殖并伴有大量细菌的肝内移位，而清除肠道细菌则肝内中性粒细胞浸润及其所伴随的炎症大幅度减轻。中性粒细胞胞外诱捕网（NETs）作为其受体，参与多种形式肝损伤，并同多种肿瘤相关。而TLR9则可能是NETs成份DNA的受体，其通路激活可导致一系列下游肿瘤相关信号通路的改变。为此，我们进行了如下实验进行研究。1. 通过构建小鼠慢性酒精摄入模型，我们发现小鼠长期酗酒时，肝内NETs形成明显增多，同时肝脏损伤以及炎症反应加重。2. 应用中性粒细胞弹性蛋白酶（NETs形成关键酶）基因缺失小鼠建立伴有慢性酒精摄入的HCC模型，结果发现NETs在酒精促进HCC发生过程中起着决定性作用，NE-/-小鼠肝脏损伤明显减轻，肿瘤发生延迟，肝内诸多炎症因子减少。3. 在建立小鼠化学诱导HCC模型的基础上联合慢性酒精摄入，同时给予肠道消毒我们发现，肠道细菌是慢性酒精摄入时NETs肝内形成的原因，清除肠道细菌后，HCC发生、肝内炎症及肿瘤相关信号通路明显抑制；4. 体内应用TLR9基因缺失小鼠构建慢性酒精摄入时的HCC模型，发现TLR9-/-小鼠NETs形成明显抑制，肿瘤发生、肝内炎症及肿瘤相关信号通路也明显减轻。综合上述研究，我们第一次提出，肠道细菌通过TLR9促进了NETs的形成，是慢性酒精摄入时HCC高发的原因。本研究的完成将为今后进一步研究酒精促HCC 发生提供方向。同时也有助于推动酗酒人群对HCC的预防，并提供新的潜在作用靶点。