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標題: | 半乳糖凝集素-3在流行性感冒病毒引發之宿主免疫反應中所扮演的角色 The Role of Galectin-3 in Influenza Virus-Induced Host Immune Responses |
作者: | Yu-Jung Chen 陳宥蓉 |
指導教授: | 劉扶東(Fu-Tong Liu) |
關鍵字: | 半乳糖凝集素-3,A型流行性感冒病毒,H5N1,免疫反應,發炎,細胞激素, Galectin-3,influenza A virus,H5N1,immune responses,inflammation,cytokines, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | A型流行性感冒病毒H5N1是一株高致病性的禽流感病毒,感染人類會引發肺炎、甚至是急性呼吸窘迫症候群,病毒所引起的細胞激素失調與促發炎細胞激素的過度產生是人類H5N1感染的致病原因之一。半乳糖凝素-3屬於動物凝集素,其共有的醣類辨識區域(carbohydrate recognition domain, CRD)可辨識具有β型半乳糖修飾的醣類。半乳糖凝素-3廣泛存在,特別是免疫與上皮細胞中,也存在於流行性感冒病毒主要的感染區域—呼吸道中。許多文獻指出半乳糖凝素-3可以調節免疫功能並且具有促進發炎的活性,也發現半乳糖凝素-3的表現與肺部損傷有關。近來研究指出外加半乳糖凝素-1和半乳糖凝素-9可抑制A型流感病毒感染,另有研究顯示內生性的半乳糖凝素-9會抑制A型流感病毒專一性的CD8 T細胞與體液免疫反應。然而,目前仍不清楚內生性的半乳糖凝素-3對於流感病毒感染的影響。
我的研究主題是探討內生性半乳糖凝素-3在H5N1流感病毒感染中的角色,特別著重於H5N1流感病毒所引起的宿主免疫反應。實驗結果顯示,在感染H5N1病毒後,半乳糖凝素-3基因剔除鼠的存活率較野生型小鼠高,表示半乳糖凝素-3基因剔除鼠較不受H5N1流感病毒感染的影響,然而,兩組小鼠中的病毒量相當。藉由蘇木素-伊紅組織染色(H&E Stain)發現半乳糖凝素-3基因剔除鼠在感染後的肺部發炎與細胞浸潤情形比較輕緩,由免疫組織染色的結果觀察到小鼠肺部中半乳糖凝素-3的表現量在感染H5N1病毒後上升,且主要由浸潤細胞表現。此外,利用流式細胞術分析浸潤細胞的組成,結果顯示受感染的半乳糖凝素-3基因剔除鼠肺部中,嗜中性白血球與巨噬細胞的比例明顯較野生型小鼠低。另外比較兩組小鼠感染後肺部中的細胞激素(cytokines),發現半乳糖凝素-3基因剔除鼠中的IL-1β濃度較低、IFN-γ濃度則較高。我們進一步利用細胞實驗探討半乳糖凝素-3對於細胞激素產生的影響,結果指出相較於控制組,取自半乳糖凝素-3基因剔除鼠的bone marrow-derived macrophages在H5N1病毒感染下釋放出較少量的促發炎細胞激素(proinflammatory cytokines) 與趨化激素(chemokines),而半乳糖凝素-3基因敲落的人類肺部上皮細胞株A549也在H5N1病毒感染下釋放出較少量的促發炎細胞激素IL-6、IFN-β與趨化激素MCP-1以及IL-8。 總結我們在探討半乳糖凝素-3於H5N1病毒感染引發之宿主免疫反應中的角色所得之實驗結果,我們推論半乳糖凝素-3可能藉由促進特定促發炎細胞激素或趨化激素的產生,以及免疫細胞的趨化,進而強化H5N1病毒的病理作用,導致嚴重肺部發炎。而半乳糖凝素-3是藉由何種機制調控細胞激素或趨化激素的產生與免疫細胞的趨化則需要更進一步的研究。此研究幫助我們了解半乳糖凝素-3在H5N1病毒致病過程中的角色,並且暗示半乳糖凝素-3也許可以發展為H5N1病毒感染引發之嚴重肺部發炎的治療標的,以期減緩過度的免疫反應。 H5N1 is a highly pathogenic strain of avian influenza virus. Human H5N1 influenza infection causes pneumonia and leads to acute respiratory distress syndrome. Virus-induced cytokine dysregulation, which is characterized by excessive production of pro-inflammatory cytokines, contributes to the pathogenesis of human H5N1 disease. Galectin-3 belongs to the galectin family, which are animal lectins recognizing β-galactoside-containing glycoconjugates by the carbohydrate recognition domains. Galectin-3 is widely distributed, particularly in immune and epithelial cells, and is also found in human airways, the primary infection site of influenza virus. Galectin-3 has been reported to regulate various immune functions and shows proinflammatory activity. Studies have indicated that galectin-3 is up-regulated in lung injury. Recently, exogenously added galectin-1 and -9 were reported to inhibit influenza A virus infection, whereas another study showed the inhibitory effect of endogenous galectin-9 on influenza A virus-specific CD8 T-cell and humoral responses. However, the function of endogenous galectin-3 in H5N1 influenza A virus infection is unclear. This project is aimed to investigate the role of endogenous galectin-3 in H5N1 influenza A virus infection, especially in H5N1-induced host immune responses. Our results showed that galectin-3 knockout (Gal-3KO) mice were less susceptible to H5N1 virus infection and had higher survival rates compared to wild-type (WT) mice. However, the viral loads were comparable between WT and Gal-3KO mice. H&E staining data indicated that Gal-3KO mice exhibited lower degrees of inflammation and neutrophil infiltrations in the lungs. By using immunohistochemistry (IHC) staining, we observed that galectin-3 was up-regulated in the lungs after H5N1 infection, and the increased galectin-3 was mainly expressed by the infiltrating cells. Besides, results from flow cytometry showed that lower frequency of macrophages and neutrophils were recruited to the lungs of infected Gal-3KO mice. In addition, lower and higher levels of IL-1β and IFN-γ were detected in the lungs of infected Gal-3KO mice, respectively. We also found that lower proinflammatory cytokines and chemokine were produced from bone marrow-derived macrophages of Gal-3KO mice compared to the WT mice during H5N1 infection. Moreover, knocking down galetcin-3 in A549 human lung epithelial cells reduced the production of proinflammatory cytokine IL-6, IFN-β and chemokines, such as MCP-1 and IL-8 in response to H5N1. Combined, our results suggest that galectin-3 may enhance the pathological effects of H5N1 virus infection by promoting the production of certain proinflammatory cytokines or chemokines, and the recruitment of immune cells, which consequently leads to more severe inflammation. Further studies are required to elucidate how endogenous galectin-3 regulates cytokine production and immune cell recruitment during H5N1 infection. Continued studies will help us understand the role of galectin-3 in H5N1 pathogenesis, and evaluate the possibility of galectin-3 as a therapeutic target for H5N1-induced severe lung inflammation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18570 |
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