【摘要】：Coxsackievirus A16(CA16)做為人類手足口病(HFMD)的重要病原體之一,以其不斷在亞洲-太平洋地區(qū)的兒童群體中所引起的HFMD大規(guī)模流行已經(jīng)成為一個(gè)重要的公眾關(guān)注對(duì)象。盡管CA16感染所導(dǎo)致的HFMD較為溫和,但已有的流行病學(xué)報(bào)道證明,CA16仍然可能在感染人群中引起少數(shù)的重癥病例,以及可能引起重復(fù)感染,因此,深入對(duì)該病原的相關(guān)研究,已經(jīng)成為一個(gè)重要的公共衛(wèi)生問(wèn)題。本論文的研究工作主要針對(duì)CA16在恒河猴感染模型中的病理學(xué)特征及免疫學(xué)反應(yīng)表現(xiàn),以及相關(guān)的CA16滅活疫苗技術(shù)原理等做了初步的探索,所得到的結(jié)果為該病毒的感染機(jī)理分析及疫苗的研究提供了初步的資料。本課題的研究工作主要分為三個(gè)部分,首先,我們?cè)谇捌诮V71感染恒河猴嬰猴模型研究的基礎(chǔ)上,在恒河猴成年猴(4～5歲)幼猴(8～月10齡)和嬰猴(1～3月齡)個(gè)體內(nèi)建立了 CA16感染動(dòng)物模型,并觀察到該病毒可通過(guò)鼻腔粘膜引起恒河猴幼猴和嬰猴出現(xiàn)與人類手足口病相似的口腔粘膜皰疹、手足部皰疹以及發(fā)熱等臨床癥狀,還可出現(xiàn)典型的病毒血癥和各組織器官病毒載量的輕微上升,其中以淋巴器官和CNS中病毒載量的上升最為明顯,同時(shí),可見(jiàn)到外周血中一過(guò)性IL-6和TNF-α的升高,這與我們?cè)诮V71恒河猴嬰猴模型時(shí)所表現(xiàn)出來(lái)的癥狀極其相似。另外,動(dòng)物器官的組織病理觀察表明,CA16的感染僅導(dǎo)致恒河猴出現(xiàn)肺臟、淋巴結(jié)等器官較輕的病理學(xué)改變,同時(shí)在皮膚皰疹病理組織中觀察到部分嗜酸性細(xì)胞的出現(xiàn)。根據(jù)CA16在臨床觀察中可能出現(xiàn)反復(fù)感染的報(bào)道,我們對(duì)已感染的恒河猴嬰猴、幼猴進(jìn)行了第二、三次的重復(fù)感染試驗(yàn),在此過(guò)程中,上述感染過(guò)程的表現(xiàn)均再次出現(xiàn)在相同動(dòng)物的身上,但這些感染均不能明顯誘導(dǎo)機(jī)體產(chǎn)生中和抗體反應(yīng),這似乎提示CA16病毒感染恒河猴、即使在重復(fù)感染的情況下,也未能誘導(dǎo)高滴度的中和抗體反應(yīng)。其次,我們的工作探討了以0、28天兩劑免疫程序的實(shí)驗(yàn)性CA16滅活病毒疫苗免疫恒河猴后的免疫反應(yīng)特征,實(shí)驗(yàn)結(jié)果表明,CA16滅活疫苗免疫恒河猴后可刺激其機(jī)體產(chǎn)生特異性細(xì)胞免疫反應(yīng),并且亦表現(xiàn)了相應(yīng)的血清中和抗體上升,但在系統(tǒng)分析其病毒攻擊后的免疫保護(hù)性和安全性的研究結(jié)果并不理想,免疫動(dòng)物在受到病毒攻擊后,85%的實(shí)驗(yàn)動(dòng)物均出現(xiàn)了典型的皰疹、病毒血癥以及相關(guān)臨床癥狀,病毒攻擊并未刺激機(jī)體血清中和抗體的四倍上升。在第三部分工作中,我們進(jìn)一步觀察表明,CA16可以直接感染DC細(xì)胞,并在其中增殖的過(guò)程中,刺激細(xì)胞上調(diào)包括IL6、IL5等細(xì)胞因子的表達(dá)。同時(shí)感染的DC細(xì)胞與T細(xì)胞孵育后,再次回輸T細(xì)胞至供體猴體內(nèi)后,仍可觀察到猴體出現(xiàn)典型的口腔及手足口部皰疹。這一結(jié)果提示CA16感染所引起的粘膜及皮膚皰疹病變,很有可能是免疫反應(yīng)的相關(guān)病理性反應(yīng)。另外,對(duì)前期人群臨床實(shí)驗(yàn)中CA16感染陽(yáng)性的血清樣品中細(xì)胞因子、趨化因子和中和抗體的分析進(jìn)一步提示了 CA16感染人體后血清中部分細(xì)胞因子及趨化因子的特征性變化。盡管本研究尚未能對(duì)CA16感染恒河猴及其免疫病理機(jī)制有全面的認(rèn)識(shí),但通過(guò)不同層次的分析所獲得的一系列研究資料,將為CA16在非人靈長(zhǎng)類的感染及免疫保護(hù)應(yīng)用的研究提供相應(yīng)的理論依據(jù),也為CA16疫苗的研發(fā)提供了參考資料。
[Abstract]:Coxsackievirus A16 (CA16) is one of the important pathogens of human hand-foot-mouth disease (HFMD), which has become an important public concern for the large-scale development of HFMD in the children's population in the Asia-Pacific region. Although the HFMD caused by the CA16 infection is mild, epidemiological studies have shown that the CA16 may still cause a small number of severe cases in the infected population, and may cause repeated infections, and therefore, in-depth study of the pathogen, It has become an important public health problem. The research work of this paper mainly focuses on the pathological characteristics and immunological reaction of CA16 in rhesus monkey infection model, and the related CA16 inactivated vaccine technical principle and so on. The results obtained provide a preliminary data for the analysis of the infection mechanism of the virus and the research of the vaccine. The research work of the subject was divided into three parts: first, we established the animal model of CA16 infection in the adult monkey (4-5 years old) and the baby (1-3 months) in the rhesus monkey (4-5 years old) and the infant (1-3 months), based on the study of the model of the EV71-infected rhesus monkey in the early stage. and observed that the virus can cause the clinical symptoms of oral mucosa herpes, hand-foot-foot-part herpes, fever and the like of the rhesus monkey and the baby monkey which can cause the rhesus monkey and the baby monkey to be caused by the nasal mucosa of the nasal cavity, and also can develop a typical viremia and a slight increase of the viral load of the tissues and organs, The most significant increase in viral load in the lymphoid organs and the CNS, while the increase in one of the peripheral blood mononuclear cells, IL-6 and TNF-1, is very similar to the symptoms we have shown in the establishment of the EV71 rhesus monkey model. In addition, the histopathological observation of animal organs showed that the infection of CA16 only resulted in the pathological changes of the lung, lymph nodes and other organs of the rhesus monkey, and the appearance of some of the eosinophils was observed in the pathological tissue of the skin. According to the report of the possible repeated infection of the CA16 in clinical observation, we conducted a second and a third repeated infection test on the infected rhesus monkey and the young monkey, in which the expression of the above-mentioned infection process again appeared on the same animal, These infections, however, do not significantly induce a neutralizing antibody reaction in the body, which appears to suggest that the CA16 virus-infected rhesus monkey, even in the case of repeated infection, fails to induce a neutralizing antibody reaction with high drop. The results show that the immune response of the CA16 inactivated vaccine to the rhesus monkey can be stimulated by the CA16 inactivated vaccine, and the immune response of the rhesus monkey can be stimulated by the CA16 inactivated vaccine. and the immune protection and the safety of the immune animals after the system analysis of the virus attack are not ideal, and after the immune animals are subjected to the virus attack, 85% of the experimental animals have typical herpes, Viremia and related clinical symptoms, viral attack did not stimulate a four-fold increase in serum and antibody levels in the body. In the third part, we have further observed that the CA16 can directly infect the DC cells and, in the course of the proliferation, stimulate the expression of cytokines including IL6, IL5, and the like. When the infected DC cells were incubated with T cells, the typical oral and hand-foot and hand-foot-mouth herpes of the monkey body could still be observed after the T-cells were returned to the donor monkey again. This result suggests that the mucosal and skin-herpes lesions caused by the infection of the CA16 are likely to be related to the pathological reaction of the immune response. In addition, the analysis of cytokines, chemokines and neutralizing antibodies in serum samples infected with CA16 in the clinical trials of the early population further showed the characteristic changes of some cytokines and chemokines in the serum of CA16 infected with the human body. Although this study has not been able to have a comprehensive understanding of the CA16 infected rhesus monkey and its immune and pathological mechanism, a series of research data obtained through different levels of analysis will provide a corresponding theoretical basis for the study of the application of CA16 in non-human primate infection and immune protection application. Reference is also made to the development of the CA16 vaccine.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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