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  • 學位論文

探討丙戊酸 (Valproic acid) 活化 Sox2 和 Oct4 promoter 的機制

The mechanism of valproic acid enhanced Sox2 and Oct4 promoter activation

指導教授 : 陳盛良
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摘要


在 2006 年,Takahashi and Yamanaka 藉由在細胞內大量表現 Oct4, Sox2, Klf4 及 c-Myc 成功將老鼠胚胎纖維母細胞誘導成具有多能性的幹細胞 (iPS cells)。 而近年研究指出 DNA methyltransferase and histone deacetylase (HDAC) inhibitors 等許多小分子化合物,可有效提升 re-programming 的效率,且有研究指出藉由加入 HDAC inhibitor: Valproic acid (VPA),可以成功的在只有大量表現 Oct4, Sox2 的情況下,產生多能性的幹細胞。 因此我們的研究集中在 VPA 這個 HDAC 的抑制劑,在我們的研究當中發現 VPA 能夠在C2C12 細胞中提升 Oct4基因的表現並增強 Oct4 1.9k promoter 的活性;在 P19E 細胞中能提升 Sox2 基因的表現,但在 Neuro-2a 和 C2C12 中則不行。 但 Sox2 promoter 的活性卻可以在 P19E 及 C2C12 細胞中被活化。 並發現在大量表現 TR2 及 PPARδ 的情況下,可以增強 VPA 活化 Oct4 promoter 的活性,表示 TR2 及 PPARδ 可能為 VPA 調控 Oct4 promoter 主要的因子。 而 VPA 對 Sox2 promoter 主要的作用位置在-2169~-2111 的區域,此區域具有 Tal1 的 binding site。 利用突變 Tal1 binding site 及 Tal1 knockdown,確實可以大幅降低 Sox2 promoter 對 VPA 的反應,證實 Tal1 對 VPA 活化 Sox2 promoter 的機制扮演重要的角色。 此外處理各種訊息傳遞抑制劑後,發現 LY294002 (Akt inhibitor) 及 rapamycin (mTOR inhibitor) 可以抑制 VPA 活化 Sox2 promoter 的活性,且 VPA 可以活化細胞內的 Akt 和 mTOR,顯示 VPA 可能藉由活化 PI3K/Akt/mTOR pathway 來活化 Sox2 及 Oct4 的 promoter。 而未來將會探討 Tal1 是否會結合在 Sox2 promoter -2169~-2111 的區域,以及瞭解 VPA 經由 PI3K/Akt/mTOR 訊息傳導路徑來影響 Sox2 promoter 活性的機制。

關鍵字

丙戊酸

並列摘要


In 2006, Takahashi and Yamanaka reprogrammed mouse embryonic fibroblasts to induced pulripotent stem cells (iPSC) by over-expressing Oct4, Sox2, Klf4 and c-Myc. In recent study, DNA methyltransferase and histone deacetylase (HDAC) inhibitors are found to improve reprogramming efficiency. And they treated valproic acid (VPA), a histone deacetylase inhibitor, to enables reprogramming of primary human fibroblasts with only two factors, Oct4 and Sox2. In this study we found VPA could enhance both the Oct4 1.9k promoter activity and Oct4 expression in C2C12. And VPA could enhance Sox2 expression in P19 cells but not in C2C12 or Neura2A cells. However, Sox2 promoter activity was induced by VPA in both C2C12 and P19 cells. In order to search for major nuclear receptor mediating VPA enhanced Oct4 promoter activation, we found that overexpressing TR2 and PPARδ could enhance the ability of VPA activate Oct4 promoter. VPA response element on Sox2 promoter was localized to the upstream region between -2169 and -2111 bp. Mutation of the Tal1-binding site in this region and Tal1 knockdown largely reduced its VPA response, implying that this binding site plays important role in this response. In addition, we found LY294002 (PI3K inhibitor) and rapamycin (mTOR inhibitor) could reduce VPA induced Sox2 promoter activity, implying that VPA may activate Oct4 and Sox2 promoters by activating PI3K/Akt/mTOR pathway. In the future, we will define whether Tal1 could directly bind to Sox2 promoter -2169~-2111 region and the mechanism by which VPA activate Sox2 promoter via PI3K/Akt/mTOR signal pathway.

並列關鍵字

Valproic acid Sox2 Oct4

參考文獻


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