【作者】 冉旭华；

【导师】 崔玉东；

【作者基本信息】 黑龙江八一农垦大学 ， 预防兽医学， 2003， 硕士

【摘要】 新城疫(Newcastle disease, ND)是由新城疫病毒(Newcastle disease virus, NDV)引起的鸡急性、高度传染性疾病。NDV是副粘病毒科(Papramyxoviridae)腮腺炎病毒属(Rubulavirus)成员。病毒核酸为单股、负链、不分节段的RNA。NDV能在鸡胚成纤维细胞(chicken embryo fibroblasts, CEF)上生长,并产生细胞病变(cytopathiceffect, CPE)。近年来的研究发现NDV感染后,除了导致组织损伤、细胞病变、细胞融合外,还能诱导宿主细胞凋亡。已经证实NDV感染后能通过干扰素和TNF途径诱导细胞凋亡,但能否通过Caspases途径诱导感染细胞凋亡尚未见报道。本研究应用不同的Caspases抑制剂Z-VAD.fmk、Z-DEVD.fmk、Z-IETD.fmk、Z-LEHD.fmk、Z-AEVD.fmk研究了参与NDV-Mukteswar株诱导CEF凋亡的Caspases,确定了NDV可以通过Caspases依赖性途径诱导CEF凋亡。同时还研究了NDV弱毒株Clone-30诱导CEF凋亡的情况。为进一步了解NDV的致病机制,将来在临床上更好地预防和治疗ND提供理论参考。为确定NDV中等毒力毒株Mukteswar诱导CEF凋亡,首先将病毒以500 TCID50的浓度感染CEF,72h后进行形态学检查和DNA片段化分析。结果NDV感染使细胞之间的连接消失,染色质浓缩断裂,细胞膜内陷,凋亡小体(apoptotic body)形成,基因组DNA断裂成188bp左右及成其整数倍的片段、琼脂糖凝胶电泳呈现典型的凋亡带,即DNA梯形电泳带(ladder)。这表明NDV能够诱导CEF凋亡。为确定病毒诱导细胞凋亡的有效浓度及细胞凋亡的时间进程,将Mukteswar株以500 TCID50的浓度感染细胞,分别提取感染后12、24、36、48、60、72 h的细胞基因组DNA,进行DNA片段化检测分析,结果在感染后48 h,DNA琼脂糖凝胶电泳出现明显的梯形电泳带。尔后,将病毒分别以100、150、200、250、300、400 TCID50的浓度感染细胞,培养48 h后进行DNA片段化检测分析,结果确定该病毒以200TCID50浓度接种为宜。以上结果表明,以200 TCID50的病毒浓度感染CEF、在感染后48 h时检测凋亡细胞的基因组DNA片段化为佳。Caspases的活化是细胞凋亡的关键。为了解在NDV诱导CEF凋亡的过程中是否有Caspases参与,在NDV-Mukteswar接种CEF前2 h用Caspases抑制剂Z-VAD处理细胞,接种病毒后进行形态学检查与DNA片段化检测分析,结果Z-VAD fmk对凋亡有明显的抑制作用,这表明NDV通过Caspases途径诱导细胞凋亡。在试验中还发现在使用抑制剂时虽然抑制了凋亡的发生,但病毒仍引起细胞病变,而且病更多还原

【Abstract】 Avian Newcastle Disease Virus (NDV) is the causative agent of chicken NewcastleDisease, an acute, highly contagious viral septic disease. Replication of NDV in chickenembryo fibroblasts (CEF) causes extensive cytopathic effect (CPE), leading to destructionof the entire cell monolayer and the death of infected cells. Now it sure that NDV caninduce apoptosis by interferon system and TNF-mediated responses. But the mechanism ofinfected cell apoptosis in chicken embryo fibroblasts is poorly documented. In this report,we show that NDV can induce CEF apoptosis. Then apoptosis of CEF induced byNDV-Mukteswar Strain was observed at 48hour post infection when 200 TCID50 was used.We show that infection of CEF with the NDV-Mukteswar strain induced caspase-dependentapoptosis. Caspase-dependent apoptosis, as characterized by chromosomal condensation,DNA fragmentation was detected in NDV-infected cells. In addition the general caspaseinhibitor Z-VAD fmk was added to the culture medium showed inhibition of the hallmarkof apoptosis at 48h postinfection. And the inhibitor of caspase-8, caspase-9 can blockedNDV-induced apoptosis too, but inhibitor of caspase-3, caspase-10 cannot inhibitNDV-induced apoptosis. At the same time, we found NDV-Clone30 strain cannot induceapoptosis in CEF. The study adds one more example to the growing list of animal virus thatinduces apoptosis through a caspase-dependent pathway.更多还原