【作者】 胡薛英；

【导师】 佘锐萍； 程国富；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2005， 博士

【摘要】 本研究运用病理形态学、临床病理学、免疫组织化学和电镜技术等多种研究方法,以新型鸭肝炎病毒人工感染9日龄健康樱桃谷雏鸭,复制新型鸭肝炎病毒人工感染疾病模型,分别在接种后12h、24h、48h、72h、96h、168h和14d共7个时间点,首次对新型鸭肝炎病毒感染过程中感染雏鸭谷丙转氨酶等13项血清生化指标的变化、组织病理学变化、病毒抗原的动态分布、NO等细胞因子的变化进行了系统的、动态观察与研究,同时重点观察了感染雏鸭肝脏和胰脏的细胞凋亡以及凋亡相关因子Caspase-3、Bcl-2、Bax、p53的动态变化。结果如下: 血清生化指标测定结果表明:血清中谷草转氨酶、谷丙转氨酶的活性升高;血清葡萄糖、总蛋白、白蛋白、球蛋白含量均表现降低;胆碱酯酶仅在接种后12h表现明显升高;血清α—淀粉酶活性无改变;碱性磷酸酶、尿酸、肌酐,钙、磷变化无明显规律;。 血液、肝和脑组织中NO的含量,TNF-α和IL-2的测定结果表明:血清中NO含量在接种后48h至接种后96h升高;肝组织中NO含量仅在接种后24h显著升高;脑组织中NO含量在整个试验期间没有变化。血清中的TNF和IL-2含量在接种后24h均表现升高,在接种后96h表现降低。 感染雏鸭的组织病理学变化结果显示:感染雏鸭的肝组织在接种后24h表现出血性坏死性肝炎,接种后48-96小时呈增生性病变;胰脏组织在接毒后24h出现胰腺细胞的局灶性坏死及嗜酸性小体,随时间推移,出现炎性细胞浸润并且逐渐增多,局灶性坏死及嗜酸性小体逐渐减少;接种后各时期脑组织病理变化呈非化脓性脑炎;脾脏表现坏死;肾小管出现坏死及异嗜性粒细胞浸润。 感染雏鸭机体内病毒抗原的动态分布结果表明:接种后不同时间,在肝脏、脾脏、胰脏、胸腺和法氏囊组织中均可检测到新型鸭肝炎病毒抗原,而肾脏、心脏、脑组织中均未检测到病毒抗原。病毒抗原均位于阳性细胞的细胞质中。 接种后48h胰脏和接种后24h肝脏的超微结构观察结果显示肝细胞和胰腺细胞出现凋亡细胞的形态特征,表现为细胞皱缩,细胞核染色质凝集边移。 TUNEL染色结果显示接种后24h至接种后96h肝脏肝细胞中有TUNEL染色阳性细胞,且在接种后24h细胞凋亡指数最高;接种后24h至接种后168h胰腺细胞中有TUNEL染色阳性细胞,在接种后24h至48h,细胞凋亡指数增加,表明肝细胞和胰腺细胞出现细胞凋亡。 感染雏鸭肝脏和胰脏组织中凋亡相关的调控因子的研究结果表明,实验感染新型鸭肝炎病毒雏鸭肝脏和胰脏组织中均可观察到caspase-3、Bcl-2、Bax阳性反应细胞,并且肝脏和胰脏中细胞凋亡指数变化方向与caspase-3、Bcl-2、Bax表达变化方向一致。在接种后12h-14d,实验感染新型鸭肝炎病毒雏鸭肝脏和胰脏组织中均未见p53阳性反应细胞,表明肝脏和胰脏中细胞凋亡与p53无关。 上述结果表明,新型鸭肝炎病毒侵入后,在肝、脾、胰、胸腺和法氏囊组织中分布,可造成感染雏鸭出现以肝出血坏死、胰局灶性坏死及嗜酸性小体形成的特征性组织病理损伤,并且出现转氨酶升高的临床特征。同时可诱导肝细胞和胰腺细胞凋亡,与凋亡相关的调控基因及细胞因子亦发生相应的变化。更多还原

【Abstract】 9-day old ducklings were inoculated with New Type Duck Hepatitis Virus. Using pathological methods, electron microscope and immunohistochemistry methods, at 12h, 24h, 48h, 72h, 168h and 14d post inoculation(PI), 13 of serum biochemical indexes, the NO of the serum, liver and brain, the TNF, IL-2 of the serum, dynamic distribution of virus, pathological changes, apoptosis, and apoptosis factor were studied.The result of 13 of serum biochemical indexes showed that: The activities of serum AST, ALT began to increased significantly at 24hPI. The levels of serum Glu, TP, ALB, GLB were dropped significantly. The activities of CHE increased significantly at 12hPI. The concentration of CRE decreased at 12h and increased at 24hPI. There were a lot of changes but have no roles in the activities of ALP, the concentration of UA, Ca and P. There were no changes in the activities of serum a-Amylase.The result of NO in the serum, liver and brain, the TNF,IL-2 in the serum showed that the levels of serum NO were increased significantly at 48h,72h and 96h post inoculation(PI). The activities of liver NO began to increased significantly at 24hPI. There were no changes in the activities of brain NO. The concentration of serum TNF and IL-2 increased significantly at 24h after inoculation, then dropped significantly at 96hPI.Histopathological examination revealed the hemorrhagic and necrotic inflammation of the liver at 24h post inoculation. At 48h to 96h post inoculation, proliferative inflammation was observed. There was limitation coagulation necrosis and Councilman’s bodies in pancreas at 24h post inoculation and it became the most extensive and serious at 48h post inoculation, between 72h to 168h post inoculation, there was heterophil infiltrating into pancreas and became more serious, the limitation coagulation necrosis and Councilman’s bodies became lighten, at 14d post inoculation, only heterophil infiltration was observed. All the time, the nonsuppurative encephalitis, necrotic splenitis was observed in the brain, spleen respectively, and it was observed heterophil infiltrating into renal leaflet.The result of dynamic distribution of virus revealed the virus were present in liver, spleen between 24hPI to 96hPI, it also present in pancreas between 24hPI to 168hPI, and between 12hPI to 14dPI, the virus were present in thymus gland and bursa Fabriicii. There were no virus present in kidney, heart and brine. It was found that the virus was located in cytoplasm of positive cells.Ultrastructural study revealed the characteristic changes of apoptosis, including cell shrinkage, chromatin condensation, were observed in liver at 24hPI and pancreas at 48hPI.The result of TUNEL staining revealed the positively cells present in liver between 24hPI to 96hPI, and apoptotic index increased to the top at 24hPI. The positively cell present in pancreas between 24hPI to 168hPI, and apoptotic index increased to the top at 48hPI.The result of immunohistochemical staining of apoptotic factor caspase-3, Bcl-2, Bax and p53 in liver and pancreas showed that, the positively cells of caspase-3, Bcl-2, Bax were observed in liver between 24hPI to 168hPI, and in pancreas from 12hPI to 14dPI, and the changes of apoptotic indexwere paralleled positively with the changes of caspase-3, Bcl-2 and Bax expression. There were no positive cells of p53 in liver and pancreas between 12hPI to 14dPI, it revealed that there was no relation between apoptosis and p53 in liver and pancreas of infected duckling with New Type Duck Hepatitis Virus.Base on the study results, it showed that New Type Duck Hepatitis Virus invaded into the body of the ducklings, distributed in organs, caused lesion of the cells. At the same time, the virus induced apoptosis of hepacytes in liver and acinar cells in pancreas, and the changes of apoptotic factor was occurred.更多还原