本文選題：腸道病毒71型(EV71) + 小鼠適應株　； 參考：《中國協(xié)和醫(yī)科大學》2010年碩士論文

【摘要】： 腸道病毒71型(Enterovirus 71, EV71)引起的腸道病毒EV71感染疾病多發(fā)生于嬰幼兒,通常引起患者手足口病和皰疹性咽峽炎,嚴重時引起無菌性腦膜炎、腦干腦炎和脊髓灰質(zhì)炎樣的麻痹等疾病,致殘及病死率較高。 自1969年發(fā)現(xiàn)以來,EV71病毒已在世界范圍內(nèi)引起十多次爆發(fā)與流行,EV71病毒在中國大陸的流行自80年代首次報道以來,已經(jīng)存在了20多年。2009年春季我國報告40,000例EV71引發(fā)的手足口病例,其中重癥病例80例。今年同期,手足口病疫情在我國又有卷土重來的趨勢,截止5月4日,全國報告手足口病例427,278例,死亡260例,其中,重癥及死亡患者以感染EV71者居多,且多為5歲以下學齡前兒童。 很多研究報道已經(jīng)證實了研制一種腸道病毒疫苗的可行性,但由于缺乏適合有效的動物模型用于評價疫苗,目前,我們?nèi)詻]有特效的藥物或是疫苗來防治EV71的感染,所以及時了解動物感染情況和建立模型,顯得非常迫切和重要。近年來,科學家通過在乳鼠和短尾猴等動物體內(nèi)進行的EV71感染實驗對病毒的致病機制、感染途徑、體內(nèi)散布等方面進行了研究,發(fā)現(xiàn)不是所有的EV71臨床分離株都能感染動物,也不是同一個EV71毒株感染不同動物出現(xiàn)的癥狀都相同,這可能是由于EV71病毒基因型和亞型的不同。所以,在本實驗中我們針對目前我國EV71方面研究的匱乏與防治藥物和疫苗研制的需要,運用阜陽地區(qū)手足口病患者臨床分離EV71株進行實驗,以期建立一個適用的EV71動物感染模型,用于EV71疫苗的研制和評價工作。 EV71病毒對動物的感染不但有物種依賴性,還有年齡依賴性。在預實驗中,我們通過對不同年齡段的ICR小鼠、SCID小鼠、豚鼠、中國恒河猴等用不同的途徑大劑量感染病毒后發(fā)現(xiàn),1日齡ICR小鼠是對本實驗室毒株最敏感的實驗動物,腹腔注射途徑是最佳的感染途徑。挑選到合適的實驗動物后,為了得到感染力更強的毒株,我們借鑒臺灣科學家復強毒株的方法,在小鼠肌肉組織連續(xù)傳代制備了EV71小鼠適應株,通過對兩毒株的比較研究發(fā)現(xiàn)：傳代株較原代株有較強的肌肉組織嗜性和RD細胞毒性；感染1日齡小鼠后在各組織中的分布更廣；在肌肉組織中的病毒量有所提高；兩毒株VP1區(qū)氨基酸系列有一定差異,可能與病毒毒力存在一定關(guān)系。利用所建立病毒檢測和模型評價體系我們對感染小鼠進行了臨床表現(xiàn)、病原學、病毒分子生物學和病理學等方面的研究,繪制出傳代株感染小鼠后7天內(nèi)病毒的復制曲線；發(fā)現(xiàn)感染后小鼠小腸、骨骼肌、心肌各組織出現(xiàn)特定病理變化；免疫組化在骨骼肌中發(fā)現(xiàn)病毒定位,從而建立起EV71小鼠感染模型,并成功運用于本研究所EV71亞單位疫苗的研制工作中 本實驗中針對EV71病毒建立的半定量RT-PCR, Real time RT-PCR, IEA, IFA,病毒分離,免疫組織化學等方法為EV71病毒的感染特點和致病機制等方面的研究提供了良好的技術(shù)基礎(chǔ),所建立的EV71感染ICR小鼠動物模型,為病毒疫苗和抗病毒藥物的研究提供了確實可用的動物評價工具。同時通過對EV71毒株的體內(nèi)、體外特性,包括細胞學和免疫學等方面特性的研究,為我國EV71病毒生物學等方面的研究也提供了實驗信息和技術(shù)支持。 綜上所述,本研究首次進行了臨床分離EV71毒株動物敏感性實驗,發(fā)現(xiàn)其能感染1日齡ICR小鼠,確定了模型的候選動物,進而針對性地制備了小鼠適應株,并建立了動物感染模型,同時建立了相應的檢測方法,尤其是實時熒光定量RT-PCR方法,并以此模型和分析體系成功評價了我所獨立研發(fā)的EV71基因亞單位疫苗。
[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 .

【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392.1

【引證文獻】

相關(guān)期刊論文 前2條

1 薛白琳;姚志紅;于榮敏;;中藥抗EV71病毒活性及其在治療手足口病中的應用[J];中國中藥雜志;2011年23期

2 王錦;許國章;;人腸道病毒71型疫苗研究進展[J];浙江預防醫(yī)學;2011年11期

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本文編號：1794977