臺灣博碩士論文加值系統

第一型單純皰疹病毒(herpes simplex virus type 1, HSV-1)是雙股螺旋DNA病毒，為一種極為常見的感染源。HSV-1感染的過程會刺激宿主細胞訊息傳遞路徑的活化，促使細胞產生第一型干擾素(type I interferon)，以抵抗病毒的入侵及擴散。目前仍不清楚HSV-1感染時對於人類口腔上皮細胞基因表現的詳細調控情形。本研究主要目的乃探討HSV-1感染人類口腔上皮細胞對其基因表現可能的調控。首先，利用基因微陣列技術(microarray)篩選出口腔上皮細胞株OEC-M1及OC3可能受到HSV-1病毒調控的基因，依照生物功能加以分類。其次，利用即時定量反轉錄酶鏈鎖反應(real time reverse transcriptase-polymerase chain reaction；real time RT-PCR)確認部分篩選出來的基因其mRNA表現於病毒感染時的變化情形。最後，利用phosphatidylinositol 3 kinase (PI3K)抑制劑(Wortmannin及LY294002)進一步探討PI3K訊息傳遞路徑和HSV-1調控細胞基因表現的關連性。研究結果發現，OEC-M1細胞與OC3細胞均因HSV-1感染而顯著上升的基因有429個，顯著下降的有357個。其中，與干擾素相關的基因：IFIT1 (interferon-induced protein with tetratricopeptide repeats 1)、IFIT2 (interferon-induced protein with tetratricopeptide repeats 2)、IFI44 (interferon-induced protein 44)、MDA5 (melanoma differentiation associated gene-5)及RIG-1 (retinoic acid-inducible gene 1)；與轉錄調控相關的FOS (FBJ murine osteosarcoma viral oncogene homolog)、MXD1 (MAX dimerization protein 1)及ATF3 (activating transcription factor 3)；及與訊息傳遞相關的DUSP6 (dual specificity phosphatase 6)及HBEGF (heparin-binding epidermal growth factor-like growth factor)，其mRNA表現量隨著HSV-1感染大致呈現上升的趨勢。此外，PI3K抑制劑能抑制HSV-1感染所造成IFIT1、IFIT2、IFI44、MDA5、RIG-1、MXD1及DUSP6表現量上升的現象；然而，PI3K抑制劑無法顯著影響因HSV-1感染所造成FOS、ATF3及HBEGF表現量上升的現象。因此，推測HSV-1感染活化PI3K可能與IFIT1、IFIT2、IFI44、MDA5、RIG-1、MXD1及DUSP6表現量的上升有關。本研究結果發現了人類口腔上皮細胞會受到HSV-1調控的基因，且部分基因的調控可能與PI3K的活化路徑有關。

Herpes simplex virus type 1(HSV-1), a double-stranded DNA virus, is one of the most common infectious agents. HSV-1 infection may activate signaling pathways of host cells, and induce the production of type I interferon to resist viral invasion. The detailed mechanisms of genes regulation in human oral epithelial cells infected by HSV-1 are not well understood. The main purpose of this study was to investigate the possible regulation of gene expression by HSV-1 in human oral epithelial cells. Microarray analysis was applied to select genes which may be regulated by HSV-1 in oral epithelial cell lines, OEC-M1 and OC3, and categorize the selected genes according to the biological functions. Transcriptional levels of several genes affected after HSV-1 infection were further validated using real time reverse transcriptase-polymerase chain reaction (real time RT-PCR). The relationship between the PI3K signaling pathway and HSV-1-regulated gene expression was examined, using the phosphatidylinositol 3 kinase (PI3K) inhibitors, Wortmannin and LY294002. In both HSV-1-infected OEC-M1 and OC3 cells, approximately 429 genes were up-regulated by HSV-1, and 357 genes were down-regulated by HSV-1. After HSV-1 infection, mRNA levels of IFN-related genes (IFIT1, IFIT2, IFI44, MDA5 and RIG-1), transcriptional regulation-related genes (FOS, MXD1 and ATF3), and signal transduction-related genes (DUSP6 and HBEGF) were up-regulated. Moreover, PI3K inhibitors, Wortmannin and LY294002 inhibited the up-regulation of IFIT1, IFIT2, IFI44, MDA5, RIG-1, MXD1 and DUSP6 by HSV-1, but failed to inhibit the HSV-1-induced expression of FOS, ATF3 and HBEGF. Therefore, the results suggested that the up-regulation of IFIT1, IFIT2, IFI44, MDA5, RIG-1, MXD1 and DUSP6 may correlate to HSV-1-activated PI3K signaling pathway. This study identified the genes regulated by HSV-1 infection in human oral epithelial cells, and discovered the regulation of several genes which may be related to PI3K activation.

頁次
目錄…………………………………………………………… I
表次目錄………………………………………………………II
圖次目錄…………………………………………………… III
中文摘要…………………………………………………… IV
英文摘要…………………………………………………… VI
導論……………………………………………………………1
材料與方法……………………………………………………8
結果………………………………………………………… 21
討論………………………………………………………… 28
表列………………………………………………………… 32
圖列………………………………………………………… 42
參考文獻…………………………………………………… 54
附錄………………………………………………………… 64

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