碩士 / 國立臺灣大學 / 生命科學系 / 103 / Fish nodavirus, the causative agent of viral nervous necrosis (VNN) of cultured marine fish, is a positive-sense single-stranded RNA virus, and will produce d.s.RNA intermediate during its replication. Fish nodavirus can stimulate innate immune responses in host cells; however, it is still unknown if melanoma different-associated protein 5 (MDA-5) can act as a d.s.RNA recognition receptor and activate intracellular innate immunity in grouper as its role in mammalian cells. In this study, poly I:C was used as an analog of viral d.s.RNA intermediate, and was transfected into a grouper brain cell line cGB which is susceptible to nervous necrosis virus. The results indicated that Poly I:C transfection could stimulate the expression of MDA-5, IRF3, IRF7 and Mx genes, and significantly increased the levels of mitochondrial oxidative substance (mROS) and autophagosomes. When MDA-5 was pre-downregulated by siRNA, the levels of Mx gene expression, mROS and autophagosomes stimulated by Poly I:C transfection all significantly decreased, indicating that d.s. RNA can induce type I interferon response, mROS and autophagy through activating MDA-5 pathway. When cGB cells were pretreated with mROS enhancer rotenone and then transfected with Poly I:C, Mx gene expression level became 3 folds higher than that in only Poly I:C-transfected cells. It is thus suggested that high mROS may synergetically enhance Poly I:C-induced Mx gene expression. Rotenone alone could upregulate autophagosome level in the treated cells, revealing that increasing of mROS could stimulate the formation of autophagosomes. When cGB cells were pretreated with mROS inhibitor and then transfected with Poly I:C, the autophagosome level decreased; furthermore, when the formation of autophagosome was inhibited by 5 mM 3MA, mROS level significantly increased, suggesting that autophagosomes may remove damaged mitochondria and downregulate mROS. This is the first report to demonstrate that MDA-5 could act as a d.s.RNA recognition receptor in fish cell line, and high mROS could synergetically enhance the Poly I:C-induced type I IFN response, and autophagy exhibited negative feedback regulation of mROS.
| Identifer | oai:union.ndltd.org:TW/103NTU05525014 |
| Date | January 2015 |
| Creators | Chun-Wei Hung, 洪焌瑋 |
| Contributors | 齊肖琪 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 60 |
Page generated in 0.018 seconds