金針菇免疫調節蛋白FIP-fve減緩呼吸道融合病毒複製和微角塵蟎發炎之研究

Inhibition of Respiratory Syncytial Virus Replication and Alleviation of Dermatophagoides Microceras-induced Inflammation by Fungal Immunomodulatory Protein FIP-Fve from Flammulina Velutipes

張語琦(Yu-Chi Chang)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學研究所/博士(2014年)

https://doi.org/10.6834/CSMU.2014.00165

摘要

FIP-fve為一個從金針菇(Flammulina velutipes)純化出來的免疫調節蛋白，具有抗發炎活性，可刺激T helper 1 (Th1)細胞激素產生，但是其對於呼吸道發炎反應之影響目前仍不清楚。感染呼吸道融合病毒(Respiratory syncytial virus , RSV)會導致幼兒細支氣管炎引發發炎反應及類氣喘症狀，呼吸道融合病毒疫苗的發展至今仍有許多尚未解決的問題。為了探討FIP-fve是否會影響呼吸道融合病毒的感染及發炎反應，我們在人類喉癌細胞(HEp-2)中處理FIP-fve並感染呼吸道融合病毒，在溶斑試驗中發現，FIP-fve可減少呼吸道融合病毒形成的空斑，接著使用免疫螢光染色以共軛焦顯微鏡觀察FIP-fve是否影響呼吸道融合病毒的吸附能力，結果發現FIP-fve並不會抑制呼吸道融合病毒吸附於細胞的能力，以西方墨點法分析呼吸道融合G蛋白，則觀察到FIP-fve可抑制呼吸道融合病毒複製。在酵素免疫分析法(ELISA)和反轉錄聚合酶鏈鎖反應(RT-PCR)中也可看到抑制呼吸道融合病毒所導致的介白素6 (interleukin 6, IL-6)表現，在human chemokine array中發現對於部分呼吸道融合病毒所刺激的趨化素，例如I-TAC、MCP-3、MIP-1α/β等也有抑制效果。在西方墨點法中也發現FIP-fve減少磷酸化IκBα 和NF-κB的蛋白表現，同時透過共軛焦顯微鏡觀察到可抑制NF-κB進入細胞核中，造成發炎相關細胞激素下降。在動物實驗中，給予Balb/c小鼠口服FIP-fve可減緩呼吸道融合病毒所刺激的呼吸道過度反應(hyperresponsiveness, AHR)、肺部發炎現象和肺部沖洗液(Bronchoalveolar lavage fluid, AHR)中IL-6的含量，而給予小鼠口服松杉靈芝(Ganodera tsugae)由益生生技公司所純化出的靈芝免疫調節蛋白FIP-gts也可看到部分相同的抑制效果。此外，家塵蟎(house dust mite, HDM)為最常見引起氣喘的原因之一，微角塵蟎(Dermatophagoides microceras, Der m)為家塵蟎之一，在台灣中部約有80%的過敏患者身上可測得微角塵蟎的抗體， FIP-fve能降低呼吸道融合病毒所導致的發炎反應和氣喘症狀，因此，我們進一步研究FIP-fve是否也能減緩微角塵蟎所造成的氣喘症狀與發炎反應。發現給予Balb/c小鼠口服FIP-fve可減緩微角塵蟎所刺激的呼吸道過度反應，以mouse cytokine array偵測小鼠肺泡沖洗液中細胞激素，發現FIP-fve可降低微角塵蟎所刺激發炎反應細胞激素像是CXCL1、MIP-1α和TNF-α產生。綜合以上實驗結果我們認為，FIP-fve可減少呼吸道融合病毒和微角塵蟎導致的發炎反應與症狀。FIP-fve是從被廣泛使用的食用性植物-金針菇所純化出的天然化合物，因此FIP-fve可能具有發展成一個安全有效的抗病毒和抗塵蟎過敏試劑之潛力。

關鍵字

免疫調節蛋白 ； 金針菇免疫調節蛋白 ； 呼吸道融合病毒 ； 塵蟎發炎反應 ； 氣喘

並列摘要

FIP-fve is an immunomodulatory protein isolated from Flammulina velutipes. It exhibits an activity of anti-inflammatory and stimulating T helper 1 (Th1) cytokine production. However, it is not clear in inhibiting airway inflammation. Respiratory syncytial virus (RSV) causes bronchiolitis in children followed by inflammation and asthma-like symptoms. To date, the development of a vaccine for this virus has been met with many problems. To determine whether FIP-fve affects the infection or consequence of immunity of RSV, we performed FIP-fve and infected RSV in HEp-2 cells. We demonstrated that FIP-fve inhibits viral titers on plaque assay. On immunofluorescence assay, FIP-fve could not decrease RSV infection. We investigated RSV G protein by Western blot, the data revealed that FIP-fve inhibited RSV replication as well as the RSV-stimulated expression of interleukin 6 (IL-6) on ELISA and RT-PCR. FIP-fve also decreased RSV-induced chemokines (I-TAC、MCP-3、MIP-1α/β) in human chemokine array. On Western blot, FIP-fve decreased both phosphorylated IκBα and NF-κB expressions. On immunofluorescence assay, FIP-fve inhibited NF-κB translocation into nucleus of A549 cells in a confocal manner. It may result in decreasing inflammatory cytokines expression. Moreover, oral FIP-fve decreased RSV-induced airway hyperresponsiveness (AHR) and IL-6 expression in BALF of Balb/c mice. Oral FIP-gts showed a similar pattern of AHR and cytokine expression from Yestern Biotech company. Asthma is a major public health concern. Its greatest risk factor is house dust mite (HDM). Dermatophagoides microceras (Der m) is a type of HDM, and in central Taiwan there is approximately 80% prevalence of sensitization to Der m. To investigate whether FIP-fve affects Der m-induced asthma and inflammation, we evaluated AHR, pathological changes, and cytokines in mice. We demonstrated that oral FIP-fve supressed Der-m-induced airway AHR, IL-6 expression and neutrophils infiltration. On mouse cytokine array, FIP-fve decreased cytokines expression (CXCL1, MIP-1α and TNF-α) in the bronchoalveolar lavage fluid (BALF) of Balb/c mice. The results of this study suggested that FIP-fve decreases RSV- and Der m-induced asthma and inflammation. FIP-fve is a natural compound from Flammulina velutipes that is widely used, and may be a safe agent for viral and allergic prevention and even therapy.

並列關鍵字

Immunomodulatory protein ； FIP-fve ； respiratory syncytial virus ； house dust mite ； inflammation ； asthma

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