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系統識別號 U0026-1807201316090900
論文名稱(中文) 人類造血幹細胞與前趨細胞非常容易受到登革病毒感染
論文名稱(英文) Human hematopoietic stem/progenitor cells are highly permissive for dengue virus infection
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 101
學期 2
出版年 102
研究生(中文) 許翊輝
研究生(英文) Yi-Huei Hsu
學號 S46984052
學位類別 碩士
語文別 英文
論文頁數 84頁
口試委員 指導教授-彭貴春
召集委員-林以行
口試委員-曾忠信
口試委員-黃效民
中文關鍵字 登革熱  幹細胞  前驅細胞  登革熱病毒  骨髓細胞  病毒型態 
英文關鍵字 Dengue  stem cells  progenitor cells  dengue virus  bone marrow cells  viral morphology 
學科別分類
中文摘要 登革熱疾病是由登革病毒所引起,在近年來除了原始流行的熱帶地區以外之地方逐漸造成對人類威脅。登革病毒屬於黃熱病毒科,它一共有四種血清型,但只就在登革病毒的自然生命週期中感染人類是關鍵且必須的。 雖然在實驗室的系統當中我們得知了許多關於登革病毒的相關知識 , 但是登革病毒仍有許多我們不清楚的特質與習性。登革主要由Aedes屬帶有登革病毒的蚊子傳播。在被蚊子叮咬之後,在發燒之前通常會有一段7到10天的潛伏期,在這個階段通常伴隨著臉部的泛紅與發熱,而在血清當中也可以偵測到NS1蛋白與登革病毒的RNA。但是病患卻一直到發燒退了才進入了危險的關鍵期,在這期間可能會發生的病兆例如:血漿外溢,休克,與器官衰竭。目前對於登革病毒沒有有效的疫苗或藥物治療而只能利用密切觀察與輔助性治療。缺乏合適的動物模型是目前登革病毒研究的重大障礙之一 , 雖然登革病毒被報導可以感染其他非人類之靈長類,但是在這些靈長類當中看不到典型的發病症狀,相反的這些動物變成無症狀的帶原者。在一個沒有動物模型的情形之下,很難得到對於登革病毒一個完整且透徹的了解,使我們更加難以詳細的了解登革病毒的致病機轉。目前一個登革病毒致病機轉之研究方向是在釐清哪一類的細胞或者哪一個特定細胞系可以被登革病毒感染。雖然在in vitro的系統當中有許多種類細胞被報導可以被登革病毒所感染;但是在in vivo當中卻沒有被證實的案例。我們目前只知道在登革病毒感染的初期,我們可以在被感染病患的周邊血液當中偵測到病毒RNA與其相關病毒蛋白。因此我們使用替代的策略: 我們試圖利用健康人類捐贈的組織或血液來進行登革病毒之感染並看是否能找出具有潛力的目標細胞。我們發現周邊血單核細胞(PBMC)幾乎不會被登革病毒所感染。但是如果捐贈者有進行GM-CSF或G-CSF的前置處理,而促使造血幹細胞從骨髓中被釋放出來到周邊血當中,這些細胞稱為周邊血幹細胞(PBSC),可以被收集獲得;我們利用登革病毒斑測驗與NS1蛋白的ELISA觀察到這些PBSC很容易被登革病毒感染。這使我們相信從骨髓放出的幹細胞與前驅細胞可能是登革熱感染的目標細胞。再者,很多臨床報告顯示在登革熱的感染早期中發現骨髓的細胞數量有低下的現象而且在發燒前夕有骨髓壓抑的現象。這些證據引領我們推測骨髓細胞與登革病毒感染有莫大關聯。因此在取得健康的新鮮人類骨髓細胞後,我們使用MOI=0.1的病毒感染。我們的結果發現以下的狀況:如同預期,登革病毒的量與NS1蛋白的表現量在被感染的人類骨髓當中遠比在PBSC當中來的高。而流式細胞儀發現有一些CD61+或CD41+的紅血球系/巨核細胞系的幹細胞或前驅細胞的子細胞群之變化百分比與病毒生產量的曲線一致,因此推斷有可能為登革病毒感染的細胞群。由CFU的測驗我們發現紅血球系與巨核細胞系在被登革病毒感染兩周後的骨髓細胞群中不存在。這證據顯示紅血球系/巨核細胞系很有可能是登革病毒初始感染的目標細胞群。另外我們發現被感染的細胞位在懸浮的細胞群當中而附著的細胞群沒有被登革病毒所感染的現象。被感染的骨髓細胞當中的病毒顆粒是在一個沒有殼體(capsid)的狀態而且其內部物質相較於傳統有殼體的VERO細胞所生產的病毒較為鬆散。我們再進一步使用了西方墨點的方式確認由骨隨細胞生產出的登革病毒沒有殼體。為了再更進一步了解病毒的型態,我們從登革熱確診但無症狀的人身上取得其血小板,進行純化之後再使用電子顯微鏡觀察其形態,發現與我們ex vivo感染的骨髓細胞所生產出的病毒型態一樣都是沒有殼體的,而且常常觀察到都與細胞膜有所關聯。綜合以上結果顯示幹細胞/骨髓細胞是在人體骨髓當中明顯的被感染族群。更重要的是,我們的結果可以突顯一些對於了解登革病毒與其疫苗發展的新穎並且關鍵之策略。
英文摘要 Dengue is induced by the infection of dengue virus and is gradually becoming a major threat to humans, causing outbreaks worldwide. Dengue virus is a member of the flavivirus family, and has four serotypes, DENV 1 to DENV 4. Even though fruitful knowledge on the biology of the virus in laboratory culture system has been obtained, many properties of dengue virus in vivo are still unanswered. After the mosquito bite, there is usually a 7 to 10-day incubation period before the onset of the fever, which is often accompanied by redness of face and detectable NS1 protein and viral RNA in the patient’s serum. But not until the regression of the fever that the critical phase in the patient begins which is often displayed by plasma leakage, shock, and organ impairment. Currently, there are no effective vaccines or antiviral drugs to prevent and treat dengue; the current practice is supportive care with intensive monitoring and observation. The pathogenesis of dengue virus infection is still hidden behind a veil till this day, despite intensive efforts and investment for decades of research. Without an animal model recapitulating the cardinal features of human dengue, it is hard to develop a full insight of dengue virus infection, thus making it more difficult to understand the detailed pathogenic cause of dengue. One of the pathogenic avenues is to investigate which cell or lineage is permissive and infectable by dengue virus. Although numerous cell lines have been reported to be permissive and infectable by dengue virus in vitro system, these cells so far have not been fully characterized in vivo. In current investigation, we observed that peripheral blood mononuclear cells (PBMC) were hardly infectable by dengue virus, while PBSCs were highly infectable by dengue virus utilizing infectious virus plaque assays and ELISA for NS1 protein in supernatants of the infected PBSC. This leads us to advocate that the stem/progenitor cells released from the bone marrow may be infectable by dengue virus infection. Interestingly, clinically, there have been many reports on the hypocellularity of the bone marrow mass during early stages of infection and bone marrow suppression prior to fever onset. Therefore, fresh human bone marrow from healthy individuals was infected with dengue virus at an MOI=0.1. Results revealed the following scenarios; the viral titer and NS1 protein produced kinetically from the infected human bone marrow was significantly higher than that from PBSC. FACS staining showed that some CD61+ or CD41+ erythroid /megakaryocyte stem/progenitor cell sub-populations percentage trend matches the peak of the virus production, thus suggesting it to be potential targets for dengue virus. With the CFU assay, the CFU-E and CFU-GEMM were not present after 2 weeks of infection. This evidence suggests the observation of these linages may be a potential target cell for dengue virus in bone marrow. Further studies revealed that the viral permissive cells were in the suspension, but not in the adherent fraction of the bone marrow cell population. The virions obtained from the supernatants of infected bone marrow appeared to be in a non-capsid configuration and were looser in its contents when compared to the virions obtained from Vero cells, which were surrounded by the classical capsid. Further confirmation with Western blot demonstrated that there was no capsid protein present in bone marrow derived virus. To further verify the observed morphology of virions, we also took platelets from asymptomatic humans with high viremia and confirmed that dengue virus in humans takes on non-capsid morphology, and is sometimes membrane associated. The cumulated results suggest that hematopoietic stem/progenitor cells are the dominant target cells infected by dengue virus in bone marrow. Importantly, our results highlight critical and new aspects for the understanding the biology of dengue virus and future vaccine approach.
論文目次 Table of Contents
Examination certificate I
Chinese Abstract II
English Abstract IV
Acknowledgments VI
Table of Contents VIII
List of Figures X
List of Tables XI
Abbreviation Index XII
Introduction 1
Materials and Methods 8
A. Materials 8
1. Cell lines 8
2. Antibodies 8
3. Reagents 11
4. Plastic and glass products 18
5. Instruments and machines 22
B. Methods 24
1. Subculture of cells 24
2. Dengue virus Plaque Assay 25
3. Patient Sample Handling 25
4. Elisa for dengue NS1 protein 26
5. Purification and isolation of dengue virus 26
6. FACS flow cytometry assay of cell samples 27
7. CFU colony forming unit assay 28
8. Electron microscope and sample preparation 29
9. Virus isolation from platelets 29
10. Western blot 30
11. Statistical analysis 31
Results
1. PBMC does not support viral replication 32
2. Erythroid/megakaryocyte progenitor cell line K562 is highly permissive for Dengue virus infection 32
3. PBSCs are highly permissive for dengue virus infection 33
4. High viral titers and high NS1 levels can be observed in Supernatants of DV infected Human BM 33
5. FACS flow cytometry analysis of infected and noninfected bone marrow cells 34
6. Proliferation BrdU assay of infected and noninfected human bone marrow 35
7. Colony forming units of erythroid and megakaryocyte (CFU-E and CFU-GEMM) do not form colonies in infected bone marrow 36
8. The suspension cell fraction of the human bone marrow is the infectable by dengue virus while cells in the adherent portion were not 37
9. Virions produced from dengue virus infected human bone marrow do not contain capsid and are highly infectious 37
10. Infectious Virus in platelets collected from dengue confirmed subject 38
Discussions 39
References 44
Figures 47
Tables 64
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