探討丙戊酸 (Valporic acid) 於肌肉細胞中活化 Oct4 promoter 的機制

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鄧涵方(Han-fang Teng) 查詢紙本館藏

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論文名稱

探討丙戊酸 (Valporic acid) 於肌肉細胞中活化 Oct4 promoter 的機制
(Defining the mechanism of valproic acid enhanced Oct4 promoter activation in myogenic cells)

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摘要(中)

多能性的胚胎幹細胞具有能夠持續生長以及分化成三個胚層細胞的能力。希望能夠利用這些幹細胞去治癒目前針對傳統醫療方式無法治癒的疾病，但其數量及種類稀少，且在來源上引起許多道德上的爭議；甚至移植於人體內會引起免疫排斥作用的干擾。Takahashi and Yamanaka 於 2006 年藉由細胞內大量表現 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特性，類似於幹細胞的 iPS cells。雖然 iPS cells 的形成開啟了利用自體細胞進行治療的可能，但仍然存在許多問題尚待解決，例如：利用病毒帶入質體的方式可能會導致插入突變的產生，以及 re-programming 的效率過低等等…。而在近年有文獻指出許多小分子化合物，像是 VPA、Bix-01924、AZA…等，可有效提升 re-programming 的效率，而我們的研究集中在 VPA 這個 HDAC 的抑制劑。在我們的研究當中發現 VPA 能夠在 P19 及 C2C12 細胞中增強 Oct4 1.9k promoter 的活性，因此希望探討其相關機制。根據實驗結果推測，VPA 可藉由徵召特定的轉錄因子至 Oct4 promoter 上活化其表現。接著進一步找出其結合位，利用 deletion 及突變 Oct4 promoter 上 hormone receptor-binding site 觀察 VPA 活化的效用是否會減弱甚至消失。而在未來的實驗當中希望能找出確切影響的因子，以及結合許多不同的小分子化合物來達到有效提升 reprogramming 效率的成果。

摘要(英)

Embryonic stem (ES) cells have the ability to grow indefinitely while maintaining pluripotency, the ability to differentiate into cells of all three germ layers. They can be used to treat a host of diseases, but there are ethical issues about their sources and problem of the possible immune rejection by the host. In 2006, Takahashi and Yamanaka successfully reprogrammed mouse embryonic fibroblasts to induced pulripotent stem cells (iPS cells) by over-expressing Oct4, Sox2, Klf4 and c-Myc. Although iPS cells open the possibility of autologous stem cell for therapy, it still has some risks, such as insertion mutagenesis and low re-programming efficiency, to be overcome. In 2008, a study showed that some small compounds, such as a histone deacetylase inhibitor called VPA, could increase the reprogramming efficiency. In our study we found that VPA could enhance the Oct4 2k promoter activity both in P19 and C2C12 cells, and it was of interest to define the mechanism. We proposed that VPA might recruit a transcription factors to the Oct4 promoter to activate its activity. We made deletion mutants of the Oct4 promoter and also mutated the hormone receptor-binding site on Oct4 promoter to see whether the VPA activaty will be abrogated. We confirmed that HRE was important in the mechanism of VPA activated the Oct4 promoter. In the future, we want to combine different small compounds to see if the iPS cells reprogramming efficiency will be increased.

關鍵字(中)

★ 誘導型多能性幹細胞
★ 肌肉細胞
★ 丙戊酸

關鍵字(英)

★ Valporic acid
★ myogenic cells
★ Induced pluripotent stem cells

論文目次

中文摘要 I
英文摘要 II
致謝 III
一、緒論 1
一、胚胎發育 (embryogenesis) 1
二、幹細胞 (stem cell) 特性 2
三、誘導型多能性幹細胞 (Induced pluripotent stem cells, iPS) 6
四、研究動機與目的 10
二、材料與方法 12
2-1-1 細胞株 12
2-2-1 質體建構 13
2-2-2 插入 (Insert) DNA 的純化 16
2-2-3 載體 (Vector) DNA 的純化 16
2-2-4接合反應 ( Ligation ) 17
2-2-5 大腸桿菌的轉型作用 (Transformation) 17
2-3 RT-PCR 17
2-4- 西方墨點實驗 (Western blot) 19
2-4-2 免疫染色 (Immunohistochemistry) 20
2-4-3 Transient promoter assay 啟動子轉染分析 21
2-4-4 Real-time PCR 22
2-4-5 藥物配置 22
三、實驗結果 23
3-1 觀察 C2C12-Retro-Py-GFP 及 C2C12-Oct4-Py-Nanog-Sox2 穩定細胞株內，經 VPA 處理後其相關基因改變情形 23
3-2 VPA 抑制 C2C12-Retro-Py-GFP 及 C2C12 的分化 24
3-3 VPA 於 C2C12 分化不同時期對其分化的影響 25
3-4 Oct4 19k promoter 於老鼠胚胎癌細胞 (P19E) 及老鼠肌纖維母細胞 (C2C12) 中，經 VPA 處理後是否會增強其活性 25
3-5 處理其它 Histone deacetylase (HDAC) inhibitor，觀察這些抑制劑是否與 VPA 同樣具有活化 Oct4 promoter 的效果 26
3-6 觀察 VPA 作用於其它 promoter 的反應 28
3-7 VPA 是否也能影響尚未嵌入染色體中的 Oct4 promoter 28
3-8 探討 VPA 作用於 Oct4 promoter 的位置 29
3-9 Hormone response element (HRE) 是否在 VPA 增強 Oct4 轉錄活性機制中，為主要的影響位置 30
3-10 Nuclear hormone receptor SF1、TR2 及 RAR 是否參與 VPA 增強 Oct4 轉錄活性的機制中 31
3-11 Nuclear hormone receptors 在 ES cell、P19、STO 及 C2C12中的表現量 32
四、實驗討論 34
4-1 VPA 與 Oct4 之間的關係 34
4-2 VPA 影響肌肉分化的情形 35
4-3 VPA 作用在 Oct4 promoter的位置 36
五、參考文獻 39
六、圖表 44
圖一、 C2C12-Retro-Py-GFP 及 C2C12-Oct4-Py-Nanog-Sox2 經 VPA 處理後其幹細胞及肌肉分化相關基因表現情形 45
圖二、細胞經處理VPA 後，其肌肉分化情形 46
圖三、細胞經處理VPA 不同時間後，其肌肉分化情形 47
圖四、 P19E 穩定表現 Oct4 promoter 細胞株對 VPA 及 RA 的反應 48
圖五、 P19E 及 C2C12 處理 VPA、TSA 及 NAM 三種不同的 Histone deacetylase (HDAC) inhibitor 後，乙醯化 H3 及 H4 蛋白質表現量差異 49
圖六、 C2C12 經高濃度 TSA 處理後其 Oct4 promoter 的反應 50
圖七、 VPA 於 C2C12 細胞中對 Pax7 50k 及 N-cadherin-42k promoter 的影響 51
圖八、 C2C12 經 transient transfection 方式於細胞內表達 pStable-Oct4 19k promoter 對 VPA 的反應，以及 RAR? 影響 VPA 對 Oct4 promoter 的效果 53
圖九、 VPA 對於不同長度 Oct4 promoter 的影響 55
圖十、 HRE 不同的 mutant type 對 VPA 的反應 57
圖十一、觀察 VPA 活化 Oct4 promoter 是否受到nuclear hormone receptor SF1、TR2 及 RAR? 的影響 59
圖十二、ES cell、P19、STO 及 C2C12 中 orphan nuclear receptros 的表現量 60
圖十三、以Q-PCR 偵測 ES cell、P19、STO 及 C2C12 中 orphan nuclear receptros 的表現量 61
附錄一 62
圖一、C2C12-Oct4-19 k-Luc 穩定細胞株對不同 HDAC inhibitors 62
以及 VPA 和 RA 的 promoter 活性 62
圖二、P19-Oct4-19 k-luc-poly 及 C2C12-Oct4-19 k-luc clone #5 對不同的 HDAC inhibitors 及 DNA demethylation agent 的 promoter 活性 63
附錄二 64
1 Primer 對照表 64
2 HDAC 抑制劑及 BIX-01294 結構圖 66
3縮寫與全名對照表 67
4溶劑及試劑配方 68

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指導教授

陳盛良(Shen-liang Chen)

審核日期

2010-7-29

推文