禽呼肠孤病毒诱导SPF鸡的细胞自噬及其与病毒复制的关系

发布时间：2018-08-08 17:35

【摘要】：禽呼肠孤病毒(Avian reovirus,ARV)是家禽的重要病原之一,其感染可引起鸡和火鸡的病毒性关节炎、腱鞘炎等,并引起免疫抑制,给养禽业造成重大的经济损失。本实验室前期已证实ARV感染能够在体外诱导细胞自噬的产生,且自噬有利于病毒的复制,并与ARV的致病机制有关。但是,ARV感染在体内是否也能够诱导细胞自噬及影响病毒的复制,还需要进一步验证。微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3, LC3/Atg8)是自噬体膜上的标记性蛋白,其表达水平在某种程度上反映了自噬的程度。本研究首先用ARVGX/2010/1毒株感染1日龄SPF鸡,并设立空白及阳性对照组,采集组织器官进行LC3蛋白检测,评价ARV感染能否诱导体内细胞自噬。结果显示,ARV感染SPF鸡48h后,即在心脏、肝脏、脾脏、肾脏、盲肠、法氏囊、胸腺等组织器官内出现了LC3-I向LC3-Ⅱ的转化,且心脏、盲肠、法氏囊的变化最为明显。进一步用透射电子显微镜观察ARV感染鸡的心脏、盲肠和法氏囊,可发现变性的线粒体和典型自噬体等超微结构的存在。上述结果说明ARV感染能够在SPF鸡体内诱导细胞自噬的发生。用ARVGX/2010/1毒株感染1日龄SPF鸡,分别于感染48h、72h、96h和120h后采取心脏、盲肠和法氏囊进行LC3蛋白检测,结果显示,感染鸡的心脏、盲肠和法氏囊中LC3-Ⅱ蛋白的表达水平随着时间的增加而增加,在72h或96h时达到高峰,96h后则递减。随后,将采取的心脏、盲肠以及法氏囊等组织处理后感染CEF细胞,用TCID50方法进行病毒滴度的检测,结果显示,心脏、盲肠和法氏囊均在72h时病毒复制水平达到高峰,96h后病毒复制水平则逐渐降低,说明ARV感染SPF鸡后诱导的自噬与病毒复制水平之间具有一定的时间依赖性,LC3-Ⅱ的表达水平和病毒复制水平均在病毒感染后72h或96h内显著升高。进一步证明,ARV感染不仅能够在鸡体内诱导自噬的发生,且能利用ARV感染触发的短暂自噬在体内进行病毒的复制与扩散,使得病毒复制水平在短时间内得到提高。本研究使用自噬抑制剂氯喹(CQ)对1日龄SPF鸡预处理后,接种ARV GX/2010/1毒株,采集心脏、盲肠和法氏囊进行LC3蛋白检测及病毒滴度的测定。结果显示,ARV感染鸡心脏内LC3-Ⅱ蛋白累积明显,而盲肠和法氏囊内LC3-Ⅱ蛋白也有所累积,表明CQ能够抑制ARV感染诱导的细胞自噬。进一步将心脏、盲肠和法氏囊处理后感染CEF细胞,用TCID50方法检测病毒滴度,结果显示,使用CQ能抑制心脏和盲肠内ARV的复制,但在法氏囊中这种现象并不明显,说明CQ能够在抑制细胞自噬的基础上抑制心脏和盲肠内的病毒复制。总之,本研究证明ARV GX/2010/1毒株感染1日龄SPF鸡后能够在体内诱导自噬的发生,但这种自噬现象是短期的,且自噬水平与病毒的复制具有一定的时间依赖性；ARV能够利用机体的短期自噬使得病毒滴度得到短暂的升高；使用自噬抑制剂CQ能够导致组织器官细胞内LC3-Ⅱ蛋白的累积,并能使得病毒复制水平略有降低。
[Abstract]:Avian reovirus (ARV) is one of the important pathogens of poultry. The infection can cause viral arthritis of chicken and Turkey, tenosynovitis and so on, and cause immunosuppression and cause great economic loss to poultry industry. This laboratory has proved that ARV infection can induce autophagy in vitro, and autophagy is beneficial to the virus. Replication is related to the pathogenesis of ARV. However, it is also necessary to further verify whether ARV infection can also induce autophagy in the body and to affect the replication of the virus. Microtubule related protein 1 light chain 3 (microtubule-associated protein 1 light chain 3, LC3/Atg8) is a marker protein on the autophagic membrane, and its expression level is in a certain range. In this study, the degree of autophagy was reflected in this study. First, 1 days old SPF chickens were infected with ARVGX/2010/1 strains and a blank and positive control group was set up. The LC3 protein of the tissues and organs was collected to evaluate whether ARV infection could induce autophagy in the body of the body. The results showed that after ARV infection of SPF chicken 48h, it was in the heart, liver, spleen, kidney, cecum, bursa, thymus. The transformation of LC3-I to LC3- II in the tissues and organs, and the most obvious changes in the heart, the cecum and the bursa of the Fabricius were observed. Further transmission electron microscopy was used to observe the heart, the cecum and the bursa of the fowl by the transmission electron microscope. The ultrastructure of the denatured mitochondria and the typical autophagic bodies could be found. The results indicated that the ARV infection could be found in the SPF chicken body. Induction of autophagy in cell autophagy. 1 days old SPF chickens were infected with ARVGX/2010/1 strains. After infection of 48h, 72h, 96h and 120h, the LC3 protein was detected in the heart, the cecum and the bursa of Fabricius. The results showed that the expression level of LC3- II protein in the heart, cecum and bursa of the infected chickens increased with the time, and reached the peak at 72h or 96h, 9. After 6h, the CEF cells were infected by the heart, the cecum and the bursa of Fabricius, and the virus titer was detected by TCID50. The results showed that the viral replication level of the heart, the cecum and the bursa of the Fabricius reached the peak at 72h, and the replication of the virus was gradually reduced after 96h, indicating the autophagy induced by ARV infected SPF chickens. There is a time dependence between the level of virus replication and the expression level of LC3- II and the level of virus replication in 72h or 96h after the virus infection. It is further proved that ARV infection can not only induce autophagy in chicken, but also use the transient autophagy triggered by ARV infection to reproduce and spread the virus in the body. The replication level of the virus was improved in a short time. In this study, the autophagic inhibitor chloroquine (CQ) was used to inoculate the ARV GX/2010/1 strain of the 1 day old SPF chicken and collect the LC3 protein and the virus titer in the heart, the cecum and the bursa of the Fabricius. The results showed that the accumulation of LC3- II protein in the heart of ARV infected chickens was obvious, and the cecum and the cecum were in the cecum and in the cecum. The LC3- II protein in the bursa was also accumulated, indicating that CQ could inhibit autophagy induced by ARV infection. CEF cells were infected after the treatment of heart, cecum and bursa, and the virus titer was detected by TCID50. The results showed that the use of CQ could inhibit the reproduction of ARV in the heart and the cecum, but the phenomenon was not obvious in the bursa of Fabricius, indicating C Q can inhibit the replication of virus in the heart and the cecum on the basis of inhibition of autophagy. In a word, this study shows that the ARV GX/2010/1 strain can induce autophagy in the body of 1 day old SPF chickens, but this autophagy phenomenon is short-term, and the level of autophagy is dependent on the replication of the virus; ARV can be used to use the machine. The short-term autophagy of the body increases the virus titer briefly, and the use of autophagy inhibitor CQ can lead to the accumulation of LC3- II protein in the tissues and organs, and can reduce the replication level of the virus slightly.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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