- 學位論文
水禽雷氏桿菌感染菜鴨肝臟初代細胞之模式建立及相關基因表現探討
Model for Riemerella anatipestifer Infecting Primary Tsaiya Duck Hepatoytes and the Following Genes Responses in Hepatocytes
摘要
水禽雷氏桿菌（Riemerella anatipestifer, RA）感染症為水禽重要疾病之一，好發於3-4週齡雛禽，可經由呼吸道及破損皮膚感染禽隻，其感染率接近100%，致死率可高達75%，造成嚴重的經濟損失。RA共有21種血清型，其中台灣以第2血清型為最常見。RA目前對於其發病機制，特別是感染後宿主的生理反應，了解甚少。本研究目的在建立一套鴨隻肝臟體外的試驗模式，並將RA感染之且進行相關實驗，提供後續研究此病之基礎。本研究使用12-20週齡褐色菜鴨（Brown Tsaiya Duck）來進行研究，以膠原蛋白酶二階段肝灌流法（collagenase two step liver perfusion）來分離鴨隻初代肝臟細胞（Primary duck hepatocytes, PDHs），成功建立出一套鴨隻體外肝臟細胞培養模式。接著使用感染複數（Multiplicity of infection, MOI）為5、10及20來攻菌0-25小時來觀察其回收率，並且在MOI=5且攻菌後16小時有最高回收率為0.1‰。接著在即時定量反轉錄聚合酶鏈式反應（Real-time quantitative reverse transcriptase PCR, qRT-PCR）實驗中，使用MOI=5攻菌後5及20小時來觀察PDHs之mRNA表現量，結果本篇研究所選擇的10個目標基因皆為向下調節基因。FGC（Fibrinogen gamma chain）與VASH1（Vasohibin）的表現量下降可能與凝血不易有關，FTH1（Ferritin, heavy polypeptide 1）、PIT54（PIT54 protein）與OTF（Ovotransferrin）的表現量下降則可能會導致肝細胞代謝功能受損，ApoA-I（Apolipoprotein A-I）與TTR（Transthyretin）則是肝細胞健康與否的指標，IL-6（Interleukin-6）與免疫有關，BRP44L（Brain protein 44-like）的表現量下降可能使細胞不易進行再生，OIH（Ovoinhibitor）則可能與產蛋率下降有關。這些基因所表現的蛋白質皆可能與RA感染症之臨床症狀有關。顯示在RA感染後，病原體與PDHs交互作用並抑制了菜鴨肝臟細胞相當程度的生理及免疫反應。另外為了觀察RA與PDHs間交互作用之形態，以MOI=40攻菌後3和6小時進行了掃描式電子顯微鏡（scanning electron microscope, SEM）試驗，並成功觀察到RA貼附以及鑲嵌在PDHs上，證實RA的感染模式主要為貼附宿主細胞後，影響細胞正常的基因表現，來達到感染及繁殖的目的。本篇研究所建立之PDHs體外試驗模式及實驗結果，可提供未來研究此病之相關幫助，對疾病的控制及疫苗開發有更多貢獻。
並列摘要
Riemerella anatipestifer (RA) infection is an important disease in waterfowl. It infects in 3-4 weeks-old ducks via the respiratory tract and damage skin. The infected rate is near 100%, and mortality is up to 75%. Twenty-one different serotypes of R. anatipestifer have been reported and serotype 2 is the major one in Taiwan. We don’t understand the pathogenesis of R. anatipestifer poorly at present, especially the physiological response of the infected cells in host. The aims of this study are that established a model of duck hepatocytes in vitro. And provide a reference for following research. The study used the 12-20 weeks-old Brown Tsaiya Duck. We used the collagenase two step liver perfusion to harvest the primary duck hepatocytes (PDHs). And that are successful. We got the recovery rates by MOI=5, 10, and 20 with bacteria for attacking in 0-25 hours. And we got the highest recovery rate to be 0.1‰ by MOI = 5 with 16 hours post infecting. We also used MOI=5 with 5, and 20 hours post infecting by real-time quantitative reverse transcriptase PCR (qRT-PCR). The results of mRNA responses from the 10 target genes in hepatocytes were all down regulation. FGC (Fibrinogen gamma chain) and VASH1 (Vasohibin) may be related to blood clotting. FTH1 (Ferritin, heavy polypeptide 1), PIT54 (PIT54 protein) and OTF (Ovotransferrin) maybe result in liver cells metabolic dysfunction. ApoA-I (Apolipoprotein AI) and TTR (Transthyretin) indicate healthy hepatocytes. IL-6 (Interleukin-6) is an immune gene. BRP44L (Brain protein 44-like) is an apoptosis gene. OIH (Ovoinhibitor) may be associated with egg production. The pathogen inhibited the physiological and immune response of the host hepatocytes. In order to observe the interaction morphology between RA and hepatocytes. We ran scanning electron microscope (SEM) test by MOI=40 with 3, and 6 hours post infecting. RA could adhere the PDHs. The infections mode of RA is that RA adhered host cell, and affected gene responses of host cells. We expect to provide a physiological response model in vitro of waterfowl in the future. It can have a contribution in the disease control and vaccine development.