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研究生:顏金彬
研究生(外文):yen jin-bin
論文名稱:CyclosporinA對黃質病毒複製之影響
論文名稱(外文):Effect of Flavivirus Infection by Cyclosporin A: Antiviral Effect of Cyclosporin A on RNA Virus Replication
指導教授:劉雨田,胡小婷廖經倫林宜玲林宜玲引用關係
指導教授(外文):Liao Ching-Len Lin Yi-Ling
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物暨免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:67
中文關鍵詞:黃質病毒複製
外文關鍵詞:AntiviralCyclosporin AFlavivirusRNA VirusReplication
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黃質病毒(Flavivirus)為具有外套膜,單股、正向的RNA病毒,而病毒複製是在宿主細胞質中所完成。Cyclosporin A(CSA)現為器官移植之抗排斥藥物,其作用標的主要是在細胞質中,因此本實驗研究CSA是否會影響黃質病毒的複製。結果顯示CSA可抑制日本腦炎病毒(Japanese Encephalitis Virus;JEV)及登革病毒(Dengue Virus;DEN)在BHK-21、N18、NT2、B2-5等細胞中複製,此抑制現象是隨CSA濃度增加而加強( 0 - 60 mg/ml );而CSA濃度高至60 mg/ml時對細胞仍無明顯毒性。CSA應不具直接毒殺病毒的能力,因在CSA直接與病毒培養後並無法抑制病毒在BHK-21細胞中形成plaque的能力。CSA非但能抑制感染細胞中病毒非結構蛋白質NS1、NS3及結構蛋白質E之表現,並可隨CSA濃度之增加而抑制日本腦炎病毒及登革病毒在BHK-21細胞中所誘發之自裁現象。由文獻報導得知CSA與FK506均可與immunophilin及calcineurin結合後,抑制細胞內calcineurin substrates之去磷酸化作用,為了進一步得知CSA的抗病毒效果是否是因為抑制calcineurin substrates之去磷酸化作用所造成,本實驗亦研究FK506對黃質病毒之影響。結果顯示FK506與CSA一樣會抑制黃質病毒之複製。另外,已知CSA與FK506均可藉與calcineurin結合而抑制phosphatase-1(calcineurin substrates之一)之去磷酸化作用,我們也探討了phosphatase-1的抑制劑如Calyculin A是否亦可抑制黃質病毒之複製;結果顯示Calyculin A在可抑制phosphatase-1的濃度下並無抑制黃質病毒之效能;此建議著CSA及FK506可能是經由將其他的受質去磷酸化而達到抑制該病毒之作用。這些現象清楚的指出CSA抑制黃質病毒與其抑制HIV的機轉不同;因為CSA是透過與HIV結構蛋白質競爭Cyclophilin A結合而抑制HIV之複製,而CSA很可能是因抑制calcineurin substrates之去磷酸化作用而造成抑制黃質病毒之複製。有趣的是CSA及FK506二種藥物無抗Sindbis virus之效果,此似建議Sindbis virus在複製時所涉之細胞成份與黃質病毒複製時所須者不同。

In cultured cell system, the antiviral effect of Cyclosporin A (CSA), an immune modulator, on Japanese encephalitis virus (JEV) and Dengue virus (DEN) was investigated in this study. Within 60 g/ml range, CSA appeared to inhibit, in a dose-dependent manner, the virus replication of JEV or DEN in baby hamster kidney (BHK-21), murine neuroblastoma N18, and human neuronal NT2 cells; in contrast, CSA had no inhibitory effect on virus replication in persistently JEV-infected B2-5 cell. Pretreatment of target cells with CSA did not enhance the antiviral effect of CSA on flaviviruses, and yet, CSA remained to be capable of inhibiting virus replication even when the late phase of virus replication was particularly sensitive to CSA inhibition. In primarily infected cells, CSA was found to profoundly reduce the intracellular accumulation of virus envelop protein, nonstructural protein-1 (NS-1) and NS-3, and restrain the extracellular release of NS-1. Moreover, CSA could block JEV- or DEN-induced apoptosis from infected cells in a dose-dependent manner. In similar experiments, the effect of FK506, which is structurally different from but functionally analogous to CSA, on JEV replication was also studied. Our results indicate the FK506, like CSA, could also abrogate JEV or DEN's replication in primarily infected cells, but not in persistently infected cells. These data suggest that the antiflaviviral effect of CSA and FK506 was likely due to their specific inhibition of calcineurin (phosphatase-2B) activity, a mechanism quite different from how CSA arrests HIV replication. However, calycurin A, which is a potent inhibitor of phosphatase-1 (one of the calcineurin substrates), failed to block flavivirus replication, indicating that CSA and FK506 might utilize yet other calcineurin substrate(s) to exert the inhibitory capability. Interestingly, neither CSA nor FK5067 could restrict the replication of Sindbis virus, an alphavirus, in cultured cells, reflecting that the antiviral effect of these two drugs was unable to apply universally to all positive-sense RNA viruses. The data herein thus provide the first molecular evidence demonstrating how immuno-suppressants CSA and FK506 could restr5ict the replication RNA viruses.

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