1型鸭甲肝病毒（DHAV-1）致雏鸭肝损伤机制的研究
摘要
鸭病毒性肝炎是由1型鸭甲肝病毒(DHAV-1)引起雏鸭的一种急性高度接触性传染病,主要侵害3周龄以内的雏鸭,特别是1周龄以内的雏鸭最易感,病死率达90%以上。虽然近两年DHAV-1的全基因序列公布于世使得DVH得到了人们的关注,国内众多研究者的注意力集中在病原学的研究,而对于DVH的发病机制的研究仍局限在上世纪的组织学和血清学水平。自然感染DHA-V与日龄有关,1-2周龄是发病的高峰期。我们前期研究已经证实4日龄的雏鸭人工感染DHAV-1后1-2天急性死亡,而35日龄的大鸭仅见一过性的精神沉郁。35日龄鸭为什么不发病,除了与免疫系统发育不完善以外,是不是与鸭肝脏发育相关,国内外对不同品种的8周龄以上鸭的肥肝发生机制研究的较多,而对于35日龄以下肉鸭肝脏脂代谢还未见报道。本课题第一部分拟对3—35日龄鸭的脂肪代谢规律进行动态的研究。其次雏鸭感染DHAV-1后快速死亡与急性肝损伤及其导致的低血糖昏迷或肝性脑病密切相关。因此本研究第二部分以临床分离的DHAV-1JX株感染动物模型,研究病毒感染后,肝脏脂质过氧化、脂代谢相关调控基因和炎症因子的改变,从而阐明鸭肝炎病毒致雏鸭肝损伤的机制。
1.3—35日龄樱桃谷肉鸭肝脏脂代谢的特点
为了弄清鸭肝炎病毒和鸭日龄的易感性差异,本研究拟在自由采食的饲养条件下,从鸭血清学和肝组织学的变化着手,动态观察35日龄内肉鸭肝脏脂肪的代谢规律。结果表明3-35肉鸭肝组织内粗脂肪的含量均大于10%,其中以3日龄肉鸭肝脏内粗脂肪含量最高,可达38.65%；肝组织冰冻切片油红O染色发现4日龄雏鸭肝脏内中性脂肪含量最高。3-35日龄肉鸭血浆脂代谢指标显示3-7日龄总胆红素(TBIL)、总胆固醇(TC)、高密度胆固醇脂(HDLC)、低密度胆固醇脂LDLC均下降,而甘油三酯(TG)升高,TBIL、TC、HDLC、LDLC、TG的变化明显的时间集中在7、14日龄。应用免疫组织化学方法对肝组织内的微粒体脂肪酸转运蛋白(MTP)、过氧化物酶增值激活受体(PPAR)和脂肪酸结合蛋白(FABP)表达进行观察：14日龄时雏鸭血浆TG升至最高,可能与组织内MTP的表达量7—14日龄下降密切相关,21日龄时肝组织中粗脂肪含量最低与该时间点的MTP的表达量上升至最高有关,暗示MTP参与肝细胞内TG的转运；PPAR在3—28日龄表达量逐渐上升,与肝组织内粗脂肪含量下降呈负相关；FABP的表达可能与肉鸭后期肝脏内TG的合成密切相关。结论：樱桃谷肉鸭1—3周龄是肝内脂质向机体其他部位输送的关键时期。
2.鸭肝炎病毒对4日龄樱桃谷肉鸭的肝损伤机制研究
禽类的肝脏是禽体内含巨噬细胞(枯否氏细胞)最多的器官,机体受到应激时,这些细胞的活化在鸭肝炎病毒感染时的作用还不清楚。鉴于雏鸭肝脏的特点,本研究从鸭肝炎病毒感染雏鸭后肝脂代谢、自由基及细胞因子等变化来阐明肝损伤的可能机制。DHAV-1JX感染雏鸭后5d,肝脏内总抗氧化能力下降,丙二醛(MDA)在感染后2-3d升高,一氧化氮(NO)及诱导性一氧化氮合酶(iNOS)在2-3d升高,引起肝脏出血和大量肝细胞变性坏死及胆管上皮的增生。荧光定量PCR检测细胞因子的转录水平：干扰素a (INFa)转录1d升高后下降,而细胞因子白细胞介素1(IL-1)、白细胞介素10(IL-10)、肿瘤坏死因子a (TNF-a)转录水平均下降,血清IL—1p感染后1d升高明显,血清IL-6变化不明显。肝细胞受损引起血液中转氨酶迅速升高,肝内MTP表达量1d急剧下降,脂代谢异常,血浆TG升高,TC降低,DLDC升高,肝脂质转运异常引发脂肪变性。DHAV-1JX致4日龄雏鸭急性肝损伤至肝功能急剧下降,血液内间接胆红素升高,血氨清除不足,血糖浓度下降,进而出现肝昏迷而快速死亡。结论：DHAV-1JX刺激雏鸭肝脏细胞因子的产生较弱,DHAV-1JX可刺激雏鸭肝脏产生大量的NO及脂类自由基。
Duck viral hepatitis (DVH) is an acute and highly contagious disease of young ducklings caused by duck hepatitis virus A1type (DHAV-1), characterized with high mortality and liver lesions included haemorrhage, fat degeneration and necrosis. Even though nearly two years whole DHAV1genome sequences got the attention of people, the domestic many researchers'attention focused on the study of etiology, and studies on the pathogenesis of DVH is still limited in the histology and serological levels. DHAV-1is the most widely distributed and can cause mortality higher than90%in ducklings in1week of age. Natural infection case was associated with age,1～2weeks is the peak of the disease.35day-old ducks had no clinical signs when they were infected with DHAV-1. why? For duck liver lipid metabolism many researchers focused in8～12week-old ducks under over feeding among different breed ducks. However, there is little information regarding lipid metabolism in any breed ducklings, and no such information is available for Cherry valley broiler ducks. To understand the possible relationship between the susceptibility to DHAV and the liver lipid content in ducklings, the age-related differences make lipid metabolisms of ducklings worthy of investigation. In this first study we examined the liver lipid metabolism by serology and liver histology under ad libitum feeding in growing broiler ducks.Moreover, The plenty of lipid may induce lipid peroxidation and free redical. Once the ducks were evoked by stress such as virus, they were apt to liver injury. So in the second part, we will focus the liver lipid peroxidation, lipid metabolism related genes and the changes of inflammatory factors in the DHAV-1animal models, so as to clarify damage mechanism of the duck hepatitis virus to duckling liver.
1. The characteristics of liver lipid metabolism in the cherry valley meat duck with3-35days
To understand the possible relationship between the susceptibility to DHAV and the liver lipid content in ducklings, the age-related differences make lipid metabolisms of ducklings worthy of investigation. The change of serology and duck liver histology, dynamic liver fat metabolism regulation were carried out within35days of age duck.
Results showed that the content of crude fat in liver tissue were greater than10%, of which3d was the highest content of crude fat in the liver, can reach38.65%. The most neutral fat of the4d liver was found in liver tissue frozen section with oil red O stainingthe plasma lipid metabolism indicators suggested total bilirubin (TBIL), total cholesterol (TC), high density lipoprotein cholesterol (HDLC) and low density lipoprotein cholesterol (LDLC) decreased from3to7days, and total glycerate (TG) increased, the time of the obvious changes was focused on7,14days of age. The microsomai triglyceride transfer protein (MTP), peroxisome proliferative activated receptor (PPAR) and fatty acid binding protein (FABP) expression in the liver were observed by immunohistochemical method:at the age of14d, the plasma TG rose to the highest, at the same time the expression of MTP decreased. Similarly, at the age of21d, the crude fat content in liver tissue was the lowest and the expression of MTP rose to the highest, which suggested that MTP were involved in TG transshipment in liver cell. PPAR expressed gradually rose and the content of crude fat rapidly declined in the liver tissue at3to28days of age, which suggested the two indexes showed a negative correlation.
FABP expression may be closely related to TG synthesis in the liver. Conclusion:1-3weeks is the key period in which intrahepatic lipid were send to other parts of the body.
2. The mechanism of duck hepatitis virus on liver damage in the4day-old cherry valley duck
Poultry liver contains most macrophages (kupffer cells), under stress, the role of the activated cells in the duck hepatitis virus infection is not clear. In view of the characteristics of duck liver, this research focused on lipid metabolism, free radical and cytokine changes to clarify the possible mechanism of liver injury. At5day post-infection (PID), the live total antioxidant capacity decreased, malonldehyde(MDA) rose2-3PID, nitric oxide (NO) and induciable nitric oxide synthase (iNOS) in2-3d increased, which caused the liver hemorrhage, degeneration necrosis and bile duct epithelial hyperplasia. Fluorescence quantitative PCR detection of cytokines transcription:INFa transcription rose at1PID and then fell, and cytokine IL-1β, IL-10, TNF-a transcription level decreased, serum IL-1β at1PID increased obviously, and serum IL-6had no significant change. Liver cell damage caused blood transaminase increasing quickly, liver MTP expression at1PID decreasing sharply, elevated serum TG, TC, DLDC, abnormal lipid transport inducing liver steatosis. DHAV-1JX caused4day-old duck acute liver damage and sharp liver dysfunction, IBIL rising in the blood, blood ammonia insufficient removal, blood sugar levels dropping, and hepatic coma. Conclusion:DHAV-1JX induced the cytokines weak production in duck liver, but can stimulate duck liver producing large amounts of NO and lipid free radicals.
1.3—35日龄樱桃谷肉鸭肝脏脂代谢的特点
为了弄清鸭肝炎病毒和鸭日龄的易感性差异,本研究拟在自由采食的饲养条件下,从鸭血清学和肝组织学的变化着手,动态观察35日龄内肉鸭肝脏脂肪的代谢规律。结果表明3-35肉鸭肝组织内粗脂肪的含量均大于10%,其中以3日龄肉鸭肝脏内粗脂肪含量最高,可达38.65%；肝组织冰冻切片油红O染色发现4日龄雏鸭肝脏内中性脂肪含量最高。3-35日龄肉鸭血浆脂代谢指标显示3-7日龄总胆红素(TBIL)、总胆固醇(TC)、高密度胆固醇脂(HDLC)、低密度胆固醇脂LDLC均下降,而甘油三酯(TG)升高,TBIL、TC、HDLC、LDLC、TG的变化明显的时间集中在7、14日龄。应用免疫组织化学方法对肝组织内的微粒体脂肪酸转运蛋白(MTP)、过氧化物酶增值激活受体(PPAR)和脂肪酸结合蛋白(FABP)表达进行观察：14日龄时雏鸭血浆TG升至最高,可能与组织内MTP的表达量7—14日龄下降密切相关,21日龄时肝组织中粗脂肪含量最低与该时间点的MTP的表达量上升至最高有关,暗示MTP参与肝细胞内TG的转运；PPAR在3—28日龄表达量逐渐上升,与肝组织内粗脂肪含量下降呈负相关；FABP的表达可能与肉鸭后期肝脏内TG的合成密切相关。结论：樱桃谷肉鸭1—3周龄是肝内脂质向机体其他部位输送的关键时期。
2.鸭肝炎病毒对4日龄樱桃谷肉鸭的肝损伤机制研究
禽类的肝脏是禽体内含巨噬细胞(枯否氏细胞)最多的器官,机体受到应激时,这些细胞的活化在鸭肝炎病毒感染时的作用还不清楚。鉴于雏鸭肝脏的特点,本研究从鸭肝炎病毒感染雏鸭后肝脂代谢、自由基及细胞因子等变化来阐明肝损伤的可能机制。DHAV-1JX感染雏鸭后5d,肝脏内总抗氧化能力下降,丙二醛(MDA)在感染后2-3d升高,一氧化氮(NO)及诱导性一氧化氮合酶(iNOS)在2-3d升高,引起肝脏出血和大量肝细胞变性坏死及胆管上皮的增生。荧光定量PCR检测细胞因子的转录水平：干扰素a (INFa)转录1d升高后下降,而细胞因子白细胞介素1(IL-1)、白细胞介素10(IL-10)、肿瘤坏死因子a (TNF-a)转录水平均下降,血清IL—1p感染后1d升高明显,血清IL-6变化不明显。肝细胞受损引起血液中转氨酶迅速升高,肝内MTP表达量1d急剧下降,脂代谢异常,血浆TG升高,TC降低,DLDC升高,肝脂质转运异常引发脂肪变性。DHAV-1JX致4日龄雏鸭急性肝损伤至肝功能急剧下降,血液内间接胆红素升高,血氨清除不足,血糖浓度下降,进而出现肝昏迷而快速死亡。结论：DHAV-1JX刺激雏鸭肝脏细胞因子的产生较弱,DHAV-1JX可刺激雏鸭肝脏产生大量的NO及脂类自由基。
Duck viral hepatitis (DVH) is an acute and highly contagious disease of young ducklings caused by duck hepatitis virus A1type (DHAV-1), characterized with high mortality and liver lesions included haemorrhage, fat degeneration and necrosis. Even though nearly two years whole DHAV1genome sequences got the attention of people, the domestic many researchers'attention focused on the study of etiology, and studies on the pathogenesis of DVH is still limited in the histology and serological levels. DHAV-1is the most widely distributed and can cause mortality higher than90%in ducklings in1week of age. Natural infection case was associated with age,1～2weeks is the peak of the disease.35day-old ducks had no clinical signs when they were infected with DHAV-1. why? For duck liver lipid metabolism many researchers focused in8～12week-old ducks under over feeding among different breed ducks. However, there is little information regarding lipid metabolism in any breed ducklings, and no such information is available for Cherry valley broiler ducks. To understand the possible relationship between the susceptibility to DHAV and the liver lipid content in ducklings, the age-related differences make lipid metabolisms of ducklings worthy of investigation. In this first study we examined the liver lipid metabolism by serology and liver histology under ad libitum feeding in growing broiler ducks.Moreover, The plenty of lipid may induce lipid peroxidation and free redical. Once the ducks were evoked by stress such as virus, they were apt to liver injury. So in the second part, we will focus the liver lipid peroxidation, lipid metabolism related genes and the changes of inflammatory factors in the DHAV-1animal models, so as to clarify damage mechanism of the duck hepatitis virus to duckling liver.
1. The characteristics of liver lipid metabolism in the cherry valley meat duck with3-35days
To understand the possible relationship between the susceptibility to DHAV and the liver lipid content in ducklings, the age-related differences make lipid metabolisms of ducklings worthy of investigation. The change of serology and duck liver histology, dynamic liver fat metabolism regulation were carried out within35days of age duck.
Results showed that the content of crude fat in liver tissue were greater than10%, of which3d was the highest content of crude fat in the liver, can reach38.65%. The most neutral fat of the4d liver was found in liver tissue frozen section with oil red O stainingthe plasma lipid metabolism indicators suggested total bilirubin (TBIL), total cholesterol (TC), high density lipoprotein cholesterol (HDLC) and low density lipoprotein cholesterol (LDLC) decreased from3to7days, and total glycerate (TG) increased, the time of the obvious changes was focused on7,14days of age. The microsomai triglyceride transfer protein (MTP), peroxisome proliferative activated receptor (PPAR) and fatty acid binding protein (FABP) expression in the liver were observed by immunohistochemical method:at the age of14d, the plasma TG rose to the highest, at the same time the expression of MTP decreased. Similarly, at the age of21d, the crude fat content in liver tissue was the lowest and the expression of MTP rose to the highest, which suggested that MTP were involved in TG transshipment in liver cell. PPAR expressed gradually rose and the content of crude fat rapidly declined in the liver tissue at3to28days of age, which suggested the two indexes showed a negative correlation.
FABP expression may be closely related to TG synthesis in the liver. Conclusion:1-3weeks is the key period in which intrahepatic lipid were send to other parts of the body.
2. The mechanism of duck hepatitis virus on liver damage in the4day-old cherry valley duck
Poultry liver contains most macrophages (kupffer cells), under stress, the role of the activated cells in the duck hepatitis virus infection is not clear. In view of the characteristics of duck liver, this research focused on lipid metabolism, free radical and cytokine changes to clarify the possible mechanism of liver injury. At5day post-infection (PID), the live total antioxidant capacity decreased, malonldehyde(MDA) rose2-3PID, nitric oxide (NO) and induciable nitric oxide synthase (iNOS) in2-3d increased, which caused the liver hemorrhage, degeneration necrosis and bile duct epithelial hyperplasia. Fluorescence quantitative PCR detection of cytokines transcription:INFa transcription rose at1PID and then fell, and cytokine IL-1β, IL-10, TNF-a transcription level decreased, serum IL-1β at1PID increased obviously, and serum IL-6had no significant change. Liver cell damage caused blood transaminase increasing quickly, liver MTP expression at1PID decreasing sharply, elevated serum TG, TC, DLDC, abnormal lipid transport inducing liver steatosis. DHAV-1JX caused4day-old duck acute liver damage and sharp liver dysfunction, IBIL rising in the blood, blood ammonia insufficient removal, blood sugar levels dropping, and hepatic coma. Conclusion:DHAV-1JX induced the cytokines weak production in duck liver, but can stimulate duck liver producing large amounts of NO and lipid free radicals.
引文
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