肠道病毒71型（EV71）小鼠模型的建立及在疫苗评价中的应用

发布时间：2018-04-24 03:32

本文选题：肠道病毒71型(EV71) + 小鼠适应株　；参考：《中国协和医科大学》2010年硕士论文

【摘要】： 肠道病毒71型(Enterovirus 71, EV71)引起的肠道病毒EV71感染疾病多发生于婴幼儿,通常引起患者手足口病和疱疹性咽峡炎,严重时引起无菌性脑膜炎、脑干脑炎和脊髓灰质炎样的麻痹等疾病,致残及病死率较高。自1969年发现以来,EV71病毒已在世界范围内引起十多次爆发与流行,EV71病毒在中国大陆的流行自80年代首次报道以来,已经存在了20多年。2009年春季我国报告40,000例EV71引发的手足口病例,其中重症病例80例。今年同期,手足口病疫情在我国又有卷土重来的趋势,截止5月4日,全国报告手足口病例427,278例,死亡260例,其中,重症及死亡患者以感染EV71者居多,且多为5岁以下学龄前儿童。很多研究报道已经证实了研制一种肠道病毒疫苗的可行性,但由于缺乏适合有效的动物模型用于评价疫苗,目前,我们仍没有特效的药物或是疫苗来防治EV71的感染,所以及时了解动物感染情况和建立模型,显得非常迫切和重要。近年来,科学家通过在乳鼠和短尾猴等动物体内进行的EV71感染实验对病毒的致病机制、感染途径、体内散布等方面进行了研究,发现不是所有的EV71临床分离株都能感染动物,也不是同一个EV71毒株感染不同动物出现的症状都相同,这可能是由于EV71病毒基因型和亚型的不同。所以,在本实验中我们针对目前我国EV71方面研究的匮乏与防治药物和疫苗研制的需要,运用阜阳地区手足口病患者临床分离EV71株进行实验,以期建立一个适用的EV71动物感染模型,用于EV71疫苗的研制和评价工作。 EV71病毒对动物的感染不但有物种依赖性,还有年龄依赖性。在预实验中,我们通过对不同年龄段的ICR小鼠、SCID小鼠、豚鼠、中国恒河猴等用不同的途径大剂量感染病毒后发现,1日龄ICR小鼠是对本实验室毒株最敏感的实验动物,腹腔注射途径是最佳的感染途径。挑选到合适的实验动物后,为了得到感染力更强的毒株,我们借鉴台湾科学家复强毒株的方法,在小鼠肌肉组织连续传代制备了EV71小鼠适应株,通过对两毒株的比较研究发现：传代株较原代株有较强的肌肉组织嗜性和RD细胞毒性；感染1日龄小鼠后在各组织中的分布更广；在肌肉组织中的病毒量有所提高；两毒株VP1区氨基酸系列有一定差异,可能与病毒毒力存在一定关系。利用所建立病毒检测和模型评价体系我们对感染小鼠进行了临床表现、病原学、病毒分子生物学和病理学等方面的研究,绘制出传代株感染小鼠后7天内病毒的复制曲线；发现感染后小鼠小肠、骨骼肌、心肌各组织出现特定病理变化；免疫组化在骨骼肌中发现病毒定位,从而建立起EV71小鼠感染模型,并成功运用于本研究所EV71亚单位疫苗的研制工作中本实验中针对EV71病毒建立的半定量RT-PCR, Real time RT-PCR, IEA, IFA,病毒分离,免疫组织化学等方法为EV71病毒的感染特点和致病机制等方面的研究提供了良好的技术基础,所建立的EV71感染ICR小鼠动物模型,为病毒疫苗和抗病毒药物的研究提供了确实可用的动物评价工具。同时通过对EV71毒株的体内、体外特性,包括细胞学和免疫学等方面特性的研究,为我国EV71病毒生物学等方面的研究也提供了实验信息和技术支持。综上所述,本研究首次进行了临床分离EV71毒株动物敏感性实验,发现其能感染1日龄ICR小鼠,确定了模型的候选动物,进而针对性地制备了小鼠适应株,并建立了动物感染模型,同时建立了相应的检测方法,尤其是实时荧光定量RT-PCR方法,并以此模型和分析体系成功评价了我所独立研发的EV71基因亚单位疫苗。
[Abstract]:Enterovirus EV71 infection caused by Enterovirus 71 ( EV71 ) occurs in infants and infants , which usually cause foot and mouth disease of the patient and herpes angina , and cause diseases such as aseptic meningitis , encephalitis and polio - like paralysis when severe , and have high disability and mortality .

Since its discovery in 1969 , EV71 has caused more than a dozen outbreaks and epidemics worldwide . The EV71 virus has been in the mainland of China for more than 20 years . In the spring of 2009 , China reported 40,000 EV71 cases of hand and foot , including 80 cases of severe cases . In the same period of this year , there were 427,278 cases of hand - foot - mouth disease in China .

In recent years , the EV71 strain of EV71 has been used to study the pathogenic mechanism , infection route and in vivo spread of EV71 .

In pre - experiment , we found that the 1 - day - old ICR mice were the most sensitive experimental animals in the laboratory .
The distribution of 1 - day - old mice was wider in the tissues .
the amount of virus in the muscle tissue is increased ;
There are some differences in amino acid sequence in VP1 region of the two strains , which may be related to viral virulence . By using the established virus detection and model evaluation system , we study the clinical manifestation , etiology , molecular biology and pathology of the infected mice , and draw the replication curve of the virus within 7 days after infected mice .
The specific pathological changes of small intestine , skeletal muscle and myocardial tissues of mice after infection were observed .
Immunohistochemical staining was found in skeletal muscle to establish an EV71 mouse infection model and successfully used in the research of EV71 subunit vaccine of the Institute .

In this experiment , we provide a good technical foundation for the study of EV71 virus infection and pathogenesis of EV71 virus by semi - quantitative RT - PCR , Real time RT - PCR , IEA , IFA , virus isolation , immunohistochemistry and so on .

In conclusion , the sensitivity experiment of EV71 strain was carried out for the first time in this study . It was found that it was able to infect ICR mice of 1 day , the candidate animals of the model were determined , and the model of animal infection was established , and the corresponding detection methods , especially real - time fluorescence quantitative RT - PCR were established , and the EV71 gene subunit vaccine was successfully evaluated by this model and analysis system .

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

【引证文献】