本文選題：腸道病毒71型 + 柯薩奇病毒A組16型�。� 參考：《廈門大學(xué)》2014年碩士論文

【摘要】：腸道病毒71型(EV71)與柯薩奇病毒A組16型(CA16)是引起兒童手足口病(HFMD)的主要病原體。近些年來,手足口病在我國的發(fā)病率逐年上升,由手足口病導(dǎo)致的重癥感染和死亡人數(shù)也明顯增加,嚴(yán)重危害了兒童的生命安全,因此急需有效的治療性藥物來治療并控制其感染與流行。單克隆抗體由于具有毒性低、特異性高等優(yōu)點(diǎn),是治療病毒性感染的良好的候選藥物。本研究的目的為優(yōu)化EV71的小鼠攻毒模型同時(shí)建立穩(wěn)定的CA16小鼠感染模型,并利用動(dòng)物模型篩選和評(píng)價(jià)具有治療潛力的EV71及CA16治療性單抗,為開展EV71及CA16治療性藥物研制提供基礎(chǔ)。 本研究首先對原有的EV71動(dòng)物模型的攻毒方式和攻毒劑量進(jìn)行了優(yōu)化,使該模型能夠達(dá)到穩(wěn)定的100%致死率。同時(shí)利用13株CA16臨床分離株對1日齡新生鼠進(jìn)行攻毒評(píng)價(jià),結(jié)果發(fā)現(xiàn)大多數(shù)CA16病毒對小鼠表現(xiàn)出了強(qiáng)毒力,攻毒小鼠表現(xiàn)出癱瘓和肢體麻痹等神經(jīng)癥狀。病理學(xué)檢測顯示攻毒小鼠的大腦和脊髓等部位出現(xiàn)了嚴(yán)重的壞死與炎癥,與CA16在人體內(nèi)的感染情況極為相似。基于上述結(jié)果,本研究建立了通過灌胃注射CA16感染1日齡BALB/c小鼠的CA16小鼠感染模型,為開展后續(xù)研究提供了重要基礎(chǔ)。 本研究應(yīng)用所建立的CA16攻毒模型對CA16中和單抗的體內(nèi)治療效果進(jìn)行評(píng)價(jià),并根據(jù)體內(nèi)外實(shí)驗(yàn)的結(jié)果確立了一株候選的CA16中和單抗14B10。通過評(píng)價(jià)感染CA16后不同治療時(shí)間點(diǎn)及治療策略的效果發(fā)現(xiàn),14B10在攻毒后2天內(nèi)進(jìn)行單針治療能夠達(dá)到超過90%的治療存活率,而利用三針治療方案則能夠在攻毒后第3天、即小鼠開始發(fā)病前1天有效治療小鼠,從而證實(shí)了14B10對CA16的感染具有良好的體內(nèi)治療能力。 本研究進(jìn)一步應(yīng)用EV71攻毒模型對13株EV71中和單抗的體內(nèi)治療能力進(jìn)行篩選評(píng)價(jià),從而確立了一株EV71中和單抗CT11F9。通過對攻毒EV71后不同治療時(shí)間和治療策略的評(píng)價(jià)發(fā)現(xiàn),CT11F9在攻毒后三天內(nèi)進(jìn)行單針治療能夠達(dá)到100%的存活率及健康率,而利用三針治療方案則能夠在攻毒后第4天、即小鼠開始發(fā)病時(shí)有效提高小鼠的存活率,且對癱瘓小鼠具有一定的治療能力。通過與人血清的阻斷ELISA實(shí)驗(yàn)發(fā)現(xiàn),CT11F9識(shí)別的表位在人血清中是免疫優(yōu)勢的。至此證實(shí)了CT11F9對EV71感染具有良好的體內(nèi)治療能力。 綜上所述,本研究優(yōu)化了原有的EV71動(dòng)物模型的攻毒參數(shù)并建立了穩(wěn)定的CA16小鼠感染模型,利用該模型成功篩選并獲得了對EV71或CA16感染具有良好治療能力的中和單抗。這些研究為研發(fā)EV71及CA16治療性抗體提供了重要的數(shù)據(jù)支持與理論基礎(chǔ)。
[Abstract]:Enterovirus 71 (EV71) and Coxsackie virus group A (CA16) are the main pathogens of HFMDs in children. In recent years, the incidence of HFMD in China has increased year by year, and the number of severe infections and deaths caused by HFMD has also increased significantly, which seriously endangers the lives of children. Therefore, effective therapeutic drugs are urgently needed to treat and control their infection and prevalence. Because of its low toxicity and high specificity, monoclonal antibody is a good candidate for the treatment of viral infection. The aim of this study was to optimize the mice model of EV71 attack and to establish a stable CA16 mouse model of infection, and to screen and evaluate the therapeutic potential of EV71 and CA16 monoclonal antibody by animal model. To provide a basis for the development of EV71 and CA16 therapeutic drugs. In this study, we first optimized the attack mode and dose of EV71 animal model, so that the model can achieve a stable mortality of 100%. At the same time, 13 clinical isolates of CA16 were used to evaluate the toxicity of 1 day old newborn mice. The results showed that most of the CA16 viruses showed strong virulence to mice, and the attacking mice showed paralysis and limb paralysis. Pathological examination showed that severe necrosis and inflammation occurred in the brain and spinal cord of the mice, which was similar to the infection of CA16 in human body. Based on the above results, the model of CA16 mice infected by intragastric injection of CA16 for 1 day in BALB/c mice was established, which provided an important basis for further study. In this study, the CA16 model was used to evaluate the therapeutic effect of CA16 neutralizing monoclonal antibody in vivo, and a candidate CA16 neutralizing monoclonal antibody 14B10 was established according to the results of in vivo and in vitro experiments. By evaluating the effects of different treatment time points and treatment strategies after CA16 infection, it was found that single needle therapy could achieve more than 90% survival rate within 2 days after drug attack, while three-needle therapy regimen could achieve the survival rate of more than 90% on the third day after drug attack. That is to say, one day before the onset of the disease, mice were effectively treated with 14B10, which proved that 14B10 had a good in vivo therapeutic ability for CA16 infection. In this study, EV71 model was used to screen and evaluate the therapeutic ability of 13 EV71 neutralizing monoclonal antibodies in vivo, and a EV71 neutralizing monoclonal antibody CT11F9 was established. Through the evaluation of different treatment time and treatment strategy after EV71 attack, it was found that the survival rate and health rate of CT11F9 could reach 100% within three days after drug attack, while the treatment with three needles could achieve 100% survival rate and health rate on the 4th day after drug attack. That is to say, the survival rate of the mice was improved effectively when the mice began to develop the disease, and the palsy mice had certain therapeutic ability. By blocking ELISA with human serum, it was found that the epitope recognized by CT11F9 was immune dominant in human serum. It has been proved that CT11F9 has good in vivo treatment of EV71 infection. To sum up, we optimized the parameters of the original EV71 animal model and established a stable CA16 mouse model of infection. Using this model, we successfully screened and obtained neutralized McAbs with good ability to treat EV71 or CA16 infection. These studies provide important data support and theoretical basis for the development of EV71 and CA16 therapeutic antibodies.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.5

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本文編號(hào)：1909708