一月龄土拨鼠肝炎病毒感染模型的建立

Establishment of animal model of woodchuck hepatitis virus infection

目的建立土拨鼠慢性病毒性肝炎的动物模型。方法对一月龄的土拨鼠进行颈外静脉注射感染土拨鼠肝炎病毒(woodchuck hepatitis virus,WHV),使用两个剂量(高剂量:2.5×107 WID50;低剂量:5×106 WID50)感染,感染后在不同时间采用荧光定量PCR法测定血清中病毒DNA拷贝数,ELISA测定血清中WHsAg和WHcAb,并监测AST和ALT,肝脏活检HE染色观察病理变化,免疫组化检测WHsAg观察肝脏内病毒分布。结果感染后16周内,土拨鼠肝脏酶学AST和ALT水平均明显升高,土拨鼠血清内病毒载量在感染后第2周开始升高,16周达到高峰期,随后,低剂量组的病毒载量开始下降,而高剂量组的病毒载量仍接近106拷贝/ml。感染动物血清内可检测到WHsAg和WHcAb,肝脏病理切片显示小灶性肝细胞坏死伴炎细胞浸润及散在出血的急性肝炎症状,胞浆内可见WHsAg阳性分布。24周时,低剂量组土拨鼠血清内WHsAg和病毒DNA无法检出,提示其为急性自限性肝炎;高剂量组土拨鼠血清内依然可检测出WHsAg和病毒DNA,提示转为慢性感染。结论建立了急性病毒性肝炎转为慢性感染的土拨鼠模型,提示急性肝炎的病程发展除与土拨鼠感染年龄相关外,也与病毒感染剂量相关。

Objective To establish a woodchuck model of hepatitis virus infection for human hepatitis B virus research.Methods Two groups of one-month-old woodchuck were infected with higher dose（2.5×107WID50）and lower dose（5×106 WID50）woodchuck hepatitis virus respectively via external jugular vein injection.Serum sample were obtained at different time points post infection for virologic and serologic analysis.The viral DNA were determined by real time PCR,WHsAg and WHcAb were detected by ELISA,and the AST and ALT activity were monitored by a biochemical analyzer.Liver tissues were sampled by biopsy for pathological and immunohistochemical examination.Results From 2wto 16 wpost infection,the activity of hepatic enzyme were higher in serum of infected-animals that of the control.The copies of viral DNA increased since 2week and reached a peak at 16 week.Then,the copies of viral DNA in the serum of lower dose virus infectedwoodchucks gradually reduced,whereas that of higher dose virus infected-woodchuck maintained higher than106copies/ml.Both WHsAg and WHcAb were detected by ELISA in serum of infected-animals.The liver of woodchuck showed focal liver cell necrosis accompanied with inflammatory cell infiltration and scattered bleeding,and WHsAg were observed in the cytoplasm of liver cell.From 16 wto 24w,the viral DNA,activity of hepatic enzyme and WHsAg were gradually reduced.In contrast,the parameters related to hepatitis virus infection were positive in high dose virus-infected animals at 24 w, which indicated that infection with lower dose virus lead to self-limited hepatitis and infection with higher dose lead to chronic hepatitis.Conclusions The one-month-old woodchucks were successfully infected with woodchuck hepatitis virus,and the model developed to chronic hepatitis after acute hepatitis when higher dose virus was used,which indicated that the progress of hepatitis was not only related to the ages of woodchucks but also affected by the dose of virus.