慢性乙型肝炎病毒感染合并非酒精性脂肪性肝病动物模型的建立与鉴定

Establishment and identification of non-alcoholic fatty liver disease in chronic hepatitis B virus infected mice

目的采用高脂饮食喂饲HBV转基因小鼠，建立慢性HBV感染合并非酒精陛脂肪性肝病（NAFLD）的动物模型。方法将携带HBV全基因组的小鼠随机分为雄性对照组、雄性模型组、雌性对照组、雌性模型组。各模型组给予高脂饮食（含胆固醇姒、猪油10％、基础饲料88％），对照组则喂饲基础饲料。分批于第8，16、24周末处死小鼠，检测体质学指标、肝肾功能、糖脂代谢等NAFLD相关指标；血清HBV分型、HBeAg、HBVDNA，以及肝组织HBsAg免疫组织化学染色等病毒学指标；并通过HE、Mason及油红O染色评价肝脏组织的病理学改变。组间均数的比较采用t检验，P〈0．05为差异有统计学意义。结果与对照组相比，不同造模时间的雌性和雄性模型组小鼠体质量、肝脏质量、肝指数均明显升高；ALT、AST、碱性磷酸酶、γ-谷氨酰转移酶、总胆红素、胆汁酸等肝功能指标受损；总胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇和空腹血糖等糖脂代谢指标也有不同程度升高。然而，各组间血清HBVDNA、HBeAg水平和肝细胞HBsAg阳性率无明显差异。组织病理学检查结果提示，造模第8周时，雌、雄模型组小鼠均可见不同程度的肝细胞脂肪变，伴小叶内散在的点状坏死及炎症细胞浸润；第24周时肝脏脂肪变及炎症虽未明显加重，但有窦周纤维化和中央静脉周围纤维化。结论成功建立慢陛HBV感染合并NAFLD动物模型，为进一步研究慢性乙型肝炎合并NAFLD发病机制、药物筛选及疗效评价等提供了可靠的实验平台。

Objective To establish and identify an animal model of non-alcoholic fatty liver disease in chronic HBV infected mice. Methods Transgenic mice with sustaining HBV production were established by microinjection of ocyte. Then they were randomly assigned into 4 groups （male control, male NAFLD model, female control and female NAFLD model） and treated with high fat diet （2% cholesterol, 10% lard, 88% forage） and common forage, respectively. NAFLD-related physical indexes, liver and kidney function, glucose and lipid metabolism were investigated at the time points of 8 weeks, 16 weeks and 24 weeks. Meanwhile, HBV type, serum levels of HBV DNA and HBeAg, immunohistochemical staining of hepatic HBsAg were detected. The establishment of NAFLD was evaluated by serum levels of total cholesterol （TC）, triglycerides, glucose, aspartate aminotransferase （AST）, alkaline phosphatase （ALP）, alanine aminotransferase （ALT）,gamma-glutamyltransferase （GGT）, etc. Histological changes were also analyzed by HE, oil red O and Masson＇s trichrome staining. The status of CHB was assessed on the basis of immunohistochemistry and real-time PCR. Results The body and liver weights, liver index in HBV transgenic mice were significantly increased in regardless of the gender of HFD feeding, and the levels of serum ALT, AST, ALP, GGT, TBIL, TBA, TC, TG, HDL-C, LDL and FBG were higher in HFD groups as compared with the control mice. Lipid droplets, cytologic ballooning and liver steatosis could be observed in most lobules of HFD groups after 8-week administration, fatty degeneration of hepatocytes, patch necrosis, mild to moderate chronic inflammatory infiltration were also observed in some of HFD feeding, reflecting the emerge of steatohepatitis. At the time point of 24-week perisinusoidal fibrosis and local fibrosis occurred in HFD groups. Immunohistochemical staining and real-time PCR analysis of the liver tissues showed positive signal of HBsAg in all groups of mice, although no significant difference was documented. Conclusion Our study suggests that animal model of NAFLD can be established in HBV transgenic mice and provide a nice animal model for further studies on NAFLD with chronic hepatitis B infection.