傳染性胰臟壞死病毒感染大眼鮭魚胚胎細胞株引發轉錄因數NF-kB之活化以造成細胞凋亡

Abstract

傳染性胰臟壞死病毒(Infectious Pancreatic Necrosis Virus, IPNV)屬於兩段雙股核醣核酸病毒科，為一感染多種經濟性魚貝類且分佈廣泛之重要水生生物病毒。根據本實驗室先前之研究，證實IPNV感染大眼鮭魚胚胎細胞株(CHSE-214)後會引發細胞凋亡。為瞭解病毒感染造成宿主細胞凋亡之調控機制，本論文欲探討轉錄因數在IPNV引發細胞凋亡所扮演之角色，尤其是已知在其他系統的細胞凋亡扮演關鍵角色之轉錄因數nuclear factor-kappa B(NF-kB)與cyclic-AMP-response-element-binding protein (CREB)。 利用electrophoretic mobility shift assay，證實轉錄因數NF-kB與CREB在病毒感染後8小時皆有明顯之活化現象；而此二轉錄因數之活化現象可為tyrosine kinase抑制劑，Genistein，所抑制，因此證實tyrosine kinases在病毒感染所造成的細胞凋亡中扮演著訊息的傳遞角色。另利用trans-activation assay，顯示NF-kB的活化可促使含NF-kB結合序列的報告基因(Luciferase)之表現，證實NF-kB之活化確可促進其下游基因之表現。進一步利用NF-kB活化抑制劑proteasome inhibitor I或II之處理，繼之以IPNV感染8小時並以Hoechst 33258與Acridine Orange染劑觀察及計數細胞凋亡比例，顯示其可從僅以IPNV感染之43.1%，降至proteasome inhibitor I或II處理後之12.5%及14.7%，證實NF-kB之活化可促進細胞之凋亡。而在西方點墨法之分析結果發現，NF-kB之活化對病毒蛋白的表現並未有促進或抑制之效果。 綜合上述結果，證實IPNV感染大眼鮭魚胚胎細胞株(CHSE-214)所造成的細胞凋亡，必須經由tyrosine kinases傳遞訊息，並進一步活化轉錄因數NF-kB而促使細胞凋亡。

Infectious Pancreatic Necrosis Virus (IPNV) belonging to the Birnaviridae is one of the widespread fish pathogen and infects many economically important finfish and shellfish. According to our previous study, IPNV infection could induce apoptosis in chinook salmon embryo cell line (CHSE-214). To clarify the regulatory mechanism in IPNV-induced apoptosis, this thesis attempted to study the host cell transcription factors, especially nuclear factor-kappa B (NF-kB) and cyclic-AMP-response-element-binding protein (CREB), which are known to play important roles in the regulation of apoptosis in other systems. As determined by electrophoretic mobility shift assays, IPNV infected CHSE-214 cells show activation of NF-kB and CREB at 8 hours post infection. A tyrosine kinase inhibitor, Genistein, prevents NF-kB and CREB activation, which suggests tyrosine kinases are key regulators in signaling pathway of IPNV-induced apoptosis. IPN V-induced activation of NF-kB DNA-binding activity correlates with the onset of NF-kB-directed expression of reporter gene in trans-activation assay. Proteasome inhibitor I and II inhibit NF-kB activation and substantially diminish IPNV-induced apoptosis from 43.1% to 12.5% and 14.7% respectively, which were quantitated by Hoechst 33258 and Acridine Orange staining. However, these two inhibitors have no influence on viral protein expression of IPNV by Western analysis. In summary, the signaling of IPNV-induced apoptosis in CHSE-214 cells is transmitted by tyrosine kinases via the activation of transcription factor NF-kB.