本文選題：腸道病毒71型(EV71) + 小鼠適應(yīng)株　； 參考：《中國(guó)協(xié)和醫(yī)科大學(xué)》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(yán)重時(shí)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(guó)大陸的流行自80年代首次報(bào)道以來,已經(jīng)存在了20多年。2009年春季我國(guó)報(bào)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(guó)又有卷土重來的趨勢(shì),截止5月4日,全國(guó)報(bào)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(xué)齡前兒童。 很多研究報(bào)道已經(jīng)證實(shí)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(dòng)物模型用于評(píng)價(jià)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(shí)了解動(dòng)物感染情況和建立模型,顯得非常迫切和重要。近年來,科學(xué)家通過在乳鼠和短尾猴等動(dòng)物體內(nèi)進(jìn)行的EV71感染實(shí)驗(yàn)對(duì)病毒的致病機(jī)制、感染途徑、體內(nèi)散布等方面進(jìn)行了研究,發(fā)現(xiàn)不是所有的EV71臨床分離株都能感染動(dòng)物,也不是同一個(gè)EV71毒株感染不同動(dòng)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(shí)驗(yàn)中我們針對(duì)目前我國(guó)EV71方面研究的匱乏與防治藥物和疫苗研制的需要,運(yùn)用阜陽(yáng)地區(qū)手足口病患者臨床分離EV71株進(jìn)行實(shí)驗(yàn),以期建立一個(gè)適用的EV71動(dòng)物感染模型,用于EV71疫苗的研制和評(píng)價(jià)工作。 EV71病毒對(duì)動(dòng)物的感染不但有物種依賴性,還有年齡依賴性。在預(yù)實(shí)驗(yàn)中,我們通過對(duì)不同年齡段的ICR小鼠、SCID小鼠、豚鼠、中國(guó)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(duì)本實(shí)驗(yàn)室毒株最敏感的實(shí)驗(yàn)動(dòng)物,腹腔注射途徑是最佳的感染途徑。挑選到合適的實(shí)驗(yàn)動(dòng)物后,為了得到感染力更強(qiáng)的毒株,我們借鑒臺(tái)灣科學(xué)家復(fù)強(qiáng)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(yīng)株,通過對(duì)兩毒株的比較研究發(fā)現(xiàn)：傳代株較原代株有較強(qiáng)的肌肉組織嗜性和RD細(xì)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(cè)和模型評(píng)價(jià)體系我們對(duì)感染小鼠進(jìn)行了臨床表現(xiàn)、病原學(xué)、病毒分子生物學(xué)和病理學(xué)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(fù)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(yùn)用于本研究所EV71亞單位疫苗的研制工作中 本實(shí)驗(yàn)中針對(duì)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(xué)等方法為EV71病毒的感染特點(diǎn)和致病機(jī)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(dòng)物模型,為病毒疫苗和抗病毒藥物的研究提供了確實(shí)可用的動(dòng)物評(píng)價(jià)工具。同時(shí)通過對(duì)EV71毒株的體內(nèi)、體外特性,包括細(xì)胞學(xué)和免疫學(xué)等方面特性的研究,為我國(guó)EV71病毒生物學(xué)等方面的研究也提供了實(shí)驗(yàn)信息和技術(shù)支持。 綜上所述,本研究首次進(jìn)行了臨床分離EV71毒株動(dòng)物敏感性實(shí)驗(yàn),發(fā)現(xiàn)其能感染1日齡ICR小鼠,確定了模型的候選動(dòng)物,進(jìn)而針對(duì)性地制備了小鼠適應(yīng)株,并建立了動(dòng)物感染模型,同時(shí)建立了相應(yīng)的檢測(cè)方法,尤其是實(shí)時(shí)熒光定量RT-PCR方法,并以此模型和分析體系成功評(píng)價(jià)了我所獨(dú)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(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1

【引證文獻(xiàn)】

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(yīng)用[J];中國(guó)中藥雜志;2011年23期

2 王錦;許國(guó)章;;人腸道病毒71型疫苗研究進(jìn)展[J];浙江預(yù)防醫(yī)學(xué);2011年11期

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本文編號(hào)：1794977