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|標題:||探討抗菌胜肽Epinecidin-1與Hepcidin 1-5影響日本青鱂魚及點帶石斑魚的免疫因子抵抗神經壞死病毒(Nervous Necrosis Virus)感染的研究|
Effects of antimicrobial peptides of Epinecidin-1 and Hepcidin 1-5 on immune response genes in Nervous Necrosis Virus infected medaka (Oryzias latipes) and grouper (Epinephelus coioides)
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|摘要:||本實驗研究抗菌胜肽Epinecidin-1以及Hepcidin 1-5，影響青鱂魚以及石斑魚的免疫因子抵抗神經壞死病毒(Nervous Necrosis Virus)的感染。
在Epinecidin-1或Hepcidin 1-5 與神經壞死病毒(Nervous Necrosis Virus)相互作用在青鱂魚以及石斑魚的研究發現，若將NNV+Epinecidin-11μg/fish混合10分鐘後注射到青鱂魚體內，其活存率可達72%。但是相同的濃度作用在石斑魚上，活存率卻只有34%。青鱂魚前後打的活存率則分別為35%與38%；但相同條件作用在石斑魚前後打的活存率則只有為25%與27%。
而以NNV+Hepcidin 1-5 10μg/fish混合10分鐘注射到青鱂魚體內，活存率可達68%。但是相同的條件濃度作用在石斑魚上，活存率卻只有34%。青鱂魚前後打的活存率則分別為35%與31%；但相同條件作用在石斑魚前後打的活存率則只有為24%與8%。
而混合施打NNV的組別，再經由31天之後再次注射NNV的結果。在NNV+Epinecidin-11μg/fish混合10分鐘的組別之中，活存率可達78%。NNV+Hepcidin 1-5 1μg/fish混合10分鐘的組別活存率可達82%。推測活存的青鱂魚已對NNV具有專一性免疫能力。而相同的條件在石斑魚中，活存率達50%( Epinecidin-1)以及53%( Hepcidin 1-5)。雖然效果較差，但是活存下來的石斑魚可能也具有專一性免疫能力。
另外由TEM的結果發現，Epinecidin-1對抗病毒的方式可能是利用破壞病毒外鞘蛋白以達到溶解病毒的效果。而Hepcidin 1-5則可能是利用包裹住病毒的方式抑制病毒與宿主接觸。更進一步利用西方墨點的方式檢測，更證明Epinecidin-1以及Hepcidin 1-5都能確實降低病毒蛋白質表現。而從組織切片的結果來看，NNV混合抗菌胜肽注射的組別不論是石斑魚或是青鱂魚感染的情況都非常的輕微甚至沒有感染的現象。而在青鱂魚以及石斑魚NNV感染眼球部分已青鱂魚感染的情形較為嚴重，但是在注射抗菌胜肽的組別上感染較NNV組別來的輕微，而石斑魚似乎受到感染的情況較青鱂魚來的低很多。另外在石斑魚腸道位置似乎有偵測到NNV感染的情況。此點在青鱂魚上並無發現。
由生物晶片的檢測結果以及定量PCR結果發現，Epinecidin-1與Hepcidin 1-5都同時會造成感染NNV的青鱂魚如IL-6ST、NFκB2、SP1、TGFB1、IFN1、SOC3、CALM3、CEBPA、β-defensin、TNF、TGFB1等基因初期的抑制，並在後期使TGFB1、TNF、IFN1等基因表現量上升。另外抗菌胜肽也會表現CYP2J2、LTB4DH此兩種基因將促使白血球間素合成，此為發炎前驅物。 SPI1會影響B淋巴球的生長。並且刺激IL-1β的分泌。
This research is about antimicrobial peptides, Epinecidin-1 and Hepcidin 1-5, which can help the immune factors of medaka(Oryzias latipes) and grouper(Epinephelus coioides) resist against Nervous Necrosis Virus (NNV). In this research, we discovered that when Epinecidin-1 or Hepcidin 1-5 interacts with NNV in medaka or grouper, the survival rate of medaka would raise to 72% by injecting the mixure of NNV and Epinecidin-1 1μg/fish into the fish ten minutes later since the mixure was created. But even we perform the same experiment on grouper, the survival rate could only be 34%. If we inject the mixture into the infected medaka, the survival rate would be 35%; however, if we inject it before the medaka gets infected, the survival rate will rise to 38%. But when it comes to grouper, the survival rate would rise only from 25% to 27%. If we inject the mixture of NNV and Epinecidin-1 1μg/fish into medaka and grouper 10 minutes later since the mixure was created, the survival rate of would rise to 68%, but that of grouper will only be 34%. The survival rates of medaka injected before and after getting infected are 35% and 31%, and those of grouper are only 34% and 8%. 31 days later, re-injecting NNV into the two groups of medaka and grouper injected with NNV+Epinecidin-1 1μg/fish and NNV+ Hepcidin 1-5 1μg/fish respectively before, we found the average survival rate rose to 78% and 82%. We believe that it is because the survived medaka already has the immunity to resist against NNV. To perform the same experiment on Grouper, we will get the results of 50 %( Epinecidin-1) and 53 %( Hepcidin 1-5), even though it is not as good as that of medaka, we believe that the survived grouper also has the immunity to resist against NNV. Besides, based on the result of transmission electron microscopy (TEM), we can figure out that Epinecidin-1 decomposes the virus by breaking its capsid, but Hepcidin 1-5 breaks the connection between the virus and the host by covering the virus. Furthermore, through western blot, we can prove that both Epinecidin-1 and Hepcidin 1-5 can actually reduce the expression of NNV protein. To view the histopathology, we can see the conditions of both the medaka and the grouper injected with the mixture of NNV+ Epinecidin-1 and NNV+ Hepcidin 1-5 are pretty minor or even without being infected. Normally the NNV infection on the eyes of medaka is much more serious than that of grouper, but this condition is less serious on the groups injected with antimicrobial peptides before, especially on grouper. In addition, we detected possible NNV infection in the intestine of grouper, but nothing was detected in that of medaka. Through the microarray analysis and realtime PCR, we found the antimicrobial peptides, Epinecidin-1 and Hepcidin 1-5, could inhibit the gene in early stage, such as IL-6ST、NFκB2、SP1、TGFB1、IFN1、SOC3、CALM3、CEBPA、β-defensin、TNF and TGFB1; otherwise, antimicrobial peptides can also increase the genetic expression in later stage, such as TGFB1、TNF and IFN1. Besides, antimicrpbial peptides could also improve the leukotriene synthesis of the two kinds of gene, CYP2J2 and LTB4DH, which are inflammation Precursors. Gene SPI1 would affect the development of cell B-lymphoid and the secretion of cytokine IL-1β. In conclusion, the primary way of Epinecidin-1 to protect medaka against NNV is to break the capsid of the virus, and that of Hepcidin 1-5 is to break the connection between the virus and the host by covering it. Both of the way can effectively reduce the number of the virus, but both of Epinecidin-1 and Hepcidin 1-5 could possibly lead to medaka’s minor immune reaction in early stage due to the decreased number of virus. Also, both of them would improve the expression of the immune gene of medaka, such as CYP2J2、LTB4DH、IL-1β and SPI1.
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