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標題: | 膽固醇於A型流感病毒感染之病毒與宿主間交互作用之影響 The Effects of Cholesterol on Virus-Host Interaction During Influenza A Virus Infection |
作者: | Shu-Chen Hsu 許書禎 |
指導教授: | 李君男(Chun-Nan Lee) |
關鍵字: | 2009年新流感病毒,膽固醇,唾液酸,血球凝集素,細胞激素, 2009 pandemic H1N1,cholesterol,sialic acid,hemagglutinin,cytokine, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 膽固醇為生物體中的重要成分,其功能包含了物質的運輸、膽鹽的形成、荷爾蒙的前驅物甚至是組成細胞膜的重要成分,並與許多慢性疾病都有相關性。膽固醇亦為脂筏的重要成分之一,其與許多病毒對於宿主細胞之吸附、進入與生長佔有重要的角色。流感病毒是一造成呼吸道感染疾病之病毒,屬於正黏液病毒屬,具有8段單股、負向RNA片段。在人類歷史中曾有多次的大流行並造成數百萬至數千萬人死亡且伴隨著巨大的經濟損失。於2009年流行之H1N1新型流感病毒亦一度造成全球大恐慌。本研究利用人類氣管上皮細胞(Calu-3)以及單核球細胞株U937所誘導之類巨噬細胞,探討高濃度膽固醇對於細胞或流感病毒之影響,以了解流感病毒與宿主細胞間之交互作用。
首先觀察膽固醇對於Calu-3與U937細胞之細胞毒性,從結果得知於0.04 mM與0.08 mM之膽固醇濃度下兩種細胞皆有80 %以上的存活率,而U937的生長曲線於高膽固醇下亦不受影響。此外,培養於高膽固醇之Calu-3細胞的形態亦沒有明顯改變。而實驗發現膽固醇會影響MTT試驗之判讀,因此本研究改以錐蟲藍排除試驗測試膽固醇之細胞毒性。於高膽固醇培養之Calu-3細胞利用蔗糖梯度離心分離細胞膜,觀察到Calu-3細胞膜中之膽固醇有顯著的增加,顯示本實驗所添加之膽固醇確實能夠提高細胞膜中之膽固醇濃度。另外,利用螢光染色的方式亦觀察到alpha 2, 3-與alpha 2, 6-鍵結之唾液酸表現量有增多的現象;但是膽固醇所造成細胞膜上之唾液酸增加之機制仍有待未來更進一步的探討。而高膽固醇細胞感染流感病毒後,表現於細胞膜上之流感病毒HA蛋白質亦有增多的現象。此外,也觀察到於高膽固醇培養出的病毒(高膽固醇病毒)於血球凝集試驗中有較強之HA結合能力;為確認實驗中兩組比較之病毒液含有等量之病毒,同時利用real-time qPCR定量病毒核酸及溶斑試驗定量具感染力的病毒,發現兩者測定之病毒量是無差異的。因此推論於高膽固醇病毒表現之HA蛋白質較對照組流感病毒(正常病毒)為多。而流感病毒於0.04 mM與0.08 mM的膽固醇濃度下在Calu-3細胞中之生長曲線並不受影響,但高膽固醇病毒相較於正常病毒在感染Calu-3細胞後有較緩慢的複製,由前人的研究結果推測,高膽固醇可能會影響病毒之苞出。在病毒密度測定的實驗中,亦發現高膽固醇病毒之密度較低,因此推測膽固醇確實會影響流感病毒之病毒顆粒。在流感病毒對於宿主細胞吸附與進入能力的試驗中,觀察到高膽固醇病毒有較強之吸附能力;因此,推測高膽固醇病毒所表現之HA蛋白質較多,以致吸附能力增強。不過,膽固醇對於流感病毒進入宿主細胞之能力並沒有觀察到顯著的影響;而先前研究指出,膽固醇與流感病毒與細胞之融合有相關性,但多於極高濃度膽固醇實驗環境下探討。所以推測因本實驗中所用之膽固醇濃度較前述研究低,而沒有觀察到病毒進入細胞之能力改變。過去研究指出膽固醇會增加細胞前發炎細胞激素的表現量,而本研究在高膽固醇細胞感染流感病毒後觀察到TNF-α、IL-6與IFN-β的mRNA表現量有減少的趨勢,此結果之意義仍有待未來更進一步的研究。 總括而論,本研究首次利用人類氣管上皮細胞並以較貼近生理濃度之膽固醇來探討膽固醇與流感病毒之關係,發現高膽固醇病毒之HA蛋白質表現量較多,且對宿主細胞之吸附能力顯著增加,但其生長能力較正常病毒緩慢而其病毒密度亦較正常病毒低。而細胞於高膽固醇環境下,細胞膜上所表現之唾液酸亦有增加的現象,且在感染流感病毒後,細胞膜上所表現之HA蛋白質亦有所增加。 Cholesterol, which involved in cellular transport, formation of bile salts, and hormone precursor, is one important component in the cell membrane and also associated with many chronic diseases such as cardiovascular disease, stroke, hypertension, and diabetes. In addition, cholesterol is also an important element of lipid rafts on cell membrane and play essential role in viral adsorption, entry, and replication. Influenza viruses belong to the family Orthomyxoviridae, the genome containing 8 segments of negative-sense single-stranded RNA, cause respiratory diseases. Influenza virus has caused many pandemics in human history and millions to tens of millions of deaths, accompanied by numerous economic losses. Recently, new pandemic H1N1 influenza virus also caused global panic in 2009. In this study, we intend to investigate the effect of cholesterol on the virus-host interaction in the human airway epithelial cell (Calu-3) and human monocyte U937 induced macrophage-like cell. First, we observed the cytotoxicity of cholesterol in Calu-3 and U937 cells. For the Calu-3 cells, over 80% of cell viability was observed under 0.08 mM of cholesterol, and the replication curve of U937 cell was also unaffected. We also found there was no change in cell morphology of Calu-3 in high cholesterol. We noted that cholesterol affected the MTT assay and therefore we used the trypan blue exclusion assay to determine the cytotoxicity of cholesterol. We also separated the cell plasma membrane of high cholesterol treated cells by sucrose gradient ultracentrifugation, and found that the concentration of cholesterol on cell plasma membrane was significantly increased. In addition, we observed the expressions of alpha-2, 3 and -2, 6 sialic acid on cell surface were increased by immunofluorescence assay, but the mechanisms need further investigation. Increased viral HA protein on cell plasma membrane was also observed in this study. Infection with similar amount of PFU and RNA copies, cholesterol-enriched virus had higher HA activity than normal influenza virus as detected by HA assay, that suggested cholesterol-enriched virus had higher HA expression on the viral surface. In this study we also observed no difference in viral growth kinetics between cholesterol treated and untreated, but cholesterol-enriched virus replicated slower than normal virus in Calu-3 cell. Moreover, the density of cholesterol-enriched virus was lower than normal virus, suggesting that cholesterol may affect influenza virus virions. In the viral adsorption and entry assay, cholesterol-enriched virus has higher adsorption ability than normal virus, but no difference in viral entry ability was observed. In previous studies, cholesterol is associated with virus fusion in extremely high cholesterol concentration. However, relatively lower cholesterol concentration was used in this study, we found that cholesterol has no effects on viral entry ability. Furthermore, we also observed the lower expression of cytokines, such as IFN-β, TNF-α, and IL-6, in high cholesterol treated cell after influenza virus infection. Previous reports indicated that cholesterol may increase the expression of proinflammatory cytokines either in vitro or in vivo. Therefore, the mechanism of lower expression of proinflammatory cytokines needs futher investigation. In conclusion, compared with normal influenza virus, more viral HA protein expression on the viral surface and higher adsorption ability, but has slower growth kinetics and lower viral density, were observed in cholesterol-enriched virus. On the other hand, high cholesterol treated cell expressed more sialic acid on cell surface, and more HA protein on the cell plasma membrane when infected with influenza virus. This is the fist investigation of the effect of cholesterol on virus-host interaction in Calu-3 cell under the condition of reasonable cholesterol concentration, and our results may provide alternative treatment strategies of influenza virus infection in patients with hypercholesterolemia in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57525 |
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顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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