膽固醇對於宿主細胞感染2009新型H1N1流感病毒之感受性研究

Abstract

肥胖已經成為全世界重要的課題，世界衛生組織預測到了2015年全球會有23億人過重，其中更有7億人達到肥胖的標準。有許多研究指出肥胖的人在感染2009新型流感病毒pandemic H1N1(pH1N1)的預後是比較差的，甚至有較高的死亡率。除此之外，也有研究發現季節流感甚至是其他感染症像是肺炎，也和肥胖有密切關係。目前有少數研究提出可能影響的原因，但是大部分都是探討肥胖對於免疫系統的影響，關於病毒學的研究卻是鮮少提及。高脂血症在肥胖是非常常見的疾病，但是高膽固醇環境對於流感病毒的影響卻依然未知。因此，本篇研究想要建立一個高膽固醇細胞培養的模式，並進而了解pH1N1感染細胞之後，病毒性質及宿主免疫反應的變化。 首先測試膽固醇對於MDCK細胞的毒性，來決定添加於培養液中的劑量。使用MTT測試細胞活性之後，在4個不同濃度膽固醇存在之下，細胞都還能維持在70%以上的存活率，最後選定0.04 mM以及0.08 mM來進行後續的實驗。當細胞在高膽固醇環境下培養24小時之後感染pH1N1病毒，觀察病毒吸附及進入細胞的能力。結果發現以膽固醇前處理培養24小時之後的細胞會使pH1N1病毒吸附能力增強，不過病毒進入細胞的效率卻變差。接著觀察病毒在高膽固醇處理的細胞對於生長的影響，發現在感染後12、24以及30小時的病毒量受到膽固醇的影響很小。但是其所產生的病毒卻有著較高的細胞結合力。除了對病毒的影響之外，也觀察在高膽固醇培養下對細胞造成的變化，以及此變化對於病毒感染後細胞激素的影響，可以發現膽固醇對於細胞的影響比病毒更為顯著。在高膽固醇培養之下，細胞表面的唾液酸明顯增加，細胞sphingosine kinase的mRNA表現量也提高。在被病毒感染之後，不論是感染後12小時或是24小時，細胞激素IFN-β、TNF-α以及IL-6的mRNA表現量都顯著提高。最後為了確定這些變化是膽固醇所造成，我們選擇了一種常見的降膽固醇藥物Lovastatin抑制膽固醇的作用以觀察是否可以反轉前述的現象，結果顯示以上所觀察到的現象都可以藉由添加Lovastatin來達到抑制的效果。 總結而言，膽固醇藉由改變細胞的組成而影響了病毒對於MDCK細胞的吸附及進入，病毒的生長雖不受膽固醇的影響，但子代病毒的細胞結合力會增加。而細胞組成的改變，可能使sphingosine kinase mRNA表現量提高，進而造成細胞被感染之後，細胞激素的mRNA表現量顯著提高。膽固醇在動物或人體内對流感病毒真正之影響，仍需更進一步之研究。

Obesity has become an important topic in the world, the World Health Organization predicted that there will be 2.3 billion people overweight on 2015, and 700 million people will surpass the obesity standard. Many studies have pointed out that obese people infected with 2009 pandemic influenza virus H1N1 (pH1N1) had poor prognosis and increased mortality. In addition, studies have found that seasonal flu or other infectious diseases like pneumonia are closely related with obesity. Only a few studies point out the condition of obesity to the outcome of infectious diseases and most of the studies were related to the impact of obesity on immune system. Scared research is focus on the virology entity. Hyperlipidemia in obesity is a very common disease, yet the impact of high cholesterol environment to the growth of influenza viruses is still unclear. Therefore, a high-cholesterol cell cultured model was established to analyze the effects on virus nature and cellular immune responses upon pandemic H1N1 infection. After enriched with high cholesterol for 24 hours, the enriched cells were infected with pH1N1, and the virus adsorption and viral entry were analyzed. First, pH1N1 virus had higher adsorption ability in cholesterol enriched cells than the virus in un-enriched cells. Nevertheless, the virus entry was decreased in cholesterol enriched cells than the virus in un-enriched cells. Similar findings were also observed in viruses generated in the cholesterol enriched cells. The effect of cholesterol on the sialic acid expression and cellular cytokines production in viruses infected cells were also investigated. The cell surface sialic acid and cellular sphingosine kinase mRNA expression were significantly increased in cells cultured under high cholesterol environment. When the cholesterol enriched cells were infected with pH1N1 viruses, the cytokine mRNA expression of IFN-β、TNF-α and IL-6 mRNA expression was significantly increased than those from virus infected , un-enriched cells. In order to confirm the above findings, a cholesterol-lowering drug Lovastatin was used to inhibit cholesterol synthesis and the effects of cholesterol on virus adsorption、entry and cytokine expression could be reversed by Lovastatin. In conclusion, cholesterol affected the virus adsorption and entry in MDCK cells by changing the composition of cells. Cholesterol did not affect the growth of the virus, but the cell binding ability in cholesterol enriched virus was increase. These results suggested that sphingosine kinase mRNA expression increased, thereby causing the cytokine mRNA expression significantly increased after infection. The association of cholesterol and the influenza virus pathogenesis in vivo needs further clarification.