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  • 學位論文

建立活體甲基化觀察系統

Establishing an In Vivo Methylation Visualization System

指導教授 : 呂昱瑋
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摘要


在哺乳類中最被廣泛研究的對位性基因修飾為去氧核醣核酸甲基化,此為一個可調控基因表現的對位性遺傳機制,且透過此機制造成的基因默化是具有可逆性的。異常的去氧核醣核酸甲基化可能會導致癌症的發生,因此,此篇論文研究動機為在活體中建立一個甲基化觀察系統,當作研究癌症初期進程的實驗平台及藥物篩選之系統。本篇論文研究方向有二,其一為去氧核醣核酸甲基化的喪失,其二為去氧核醣核酸甲基化的獲增。首先,去氧核醣核酸甲基化的喪失,有許多對位性基因修飾相關的藥物都已被證實,能將癌症細胞中高度去氧核醣核酸甲基化的形式,逆轉成低度去氧核醣核酸甲基化的形式,可重新激活腫瘤抑制基因的表現,進而抑制癌細胞的生長;透過對位性基因修飾相關的藥物造成去氧核醣核酸甲基化的喪失,並研究去氧核醣核酸甲基化的喪失對於腫瘤生長速率之影響。此外,已有報導顯示和ENSA功能拮抗的五種糖尿病用藥,對於癌症細胞株的生長具有抑制之效果,並在之前的研究中透過即時半定量甲基化特異性聚合酶連鎖反應發現,ENSA在早期肝癌病人中普遍呈現低度甲基化的現象,因此,利用實驗室已申請專利的Targeted DNA Methylation (TDM),將ENSA啟動子受到甲基化修飾,其為去氧核醣核酸甲基化的獲得,利用裸鼠觀察ENSA在肝癌初期的角色。本篇論文中,發現ENSA啟動子去甲基化的發生,會使腫瘤生長速率較為緩慢,這與先前文獻在造血系統的癌症細胞實驗中處理5-Aza觀察到的現象相符,雖然尚未釐清ENSA啟動子發生甲基化時,對於腫瘤成長速率之影響為何。但已初步建立了一套活體甲基化觀察系統,能夠在活體中即時的觀測特定基因啟動子片段甲基化的程度,未來可能透過此系統在活體中篩檢去甲基化的藥物。

並列摘要


DNA methylation is an epigenetic mechanism that governs gene silencing and this silencing can be reversed. Abnormal DNA methylation pattern can lead to tumorgenesis. Therefore, we aim to develop an in vivo methylation visualization system to monitor the tumor progression. We establish a two-component platform that can be used to visulize the loss of DNA methylation or the gain of DNA methylation. First, for the loss of DNA methylation, an in vivo monitoring system is employed to observe near-infared fluorescence protein signal by FMT. 5-aza-2’-deoxycytidine is a nucleoside analog, DNA methylation inhibitor that has been shown to inhibit the activity of DNA methyltransferase and modulate the epigenetic regulation by reversing methylation in CpG islands. Second, for the gain of DNA methylation we use Targeted DNA Methylation(TDM) method to methylate the ENSA promoter, ENSA was found to be critical for liver cancer initiation. In combination, we have established a global methylation visualization system.

參考文獻


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