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研究生： 黃凱民
Kai-min Huang
論文名稱： 在終極肌肉分化時，肌肉性bHLH轉錄因子對PGC-1α的調控
Myogenic basic helix-loop-helix transcription factors regulate PGC-1α during terminal myogenic differentiation
指導教授： 陳盛良
Shen-Liang Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 生醫理工學院 - 生命科學系
Department of Life Science
畢業學年度： 95
語文別： 中文
論文頁數： 88
中文關鍵詞： 過氧化物酶增殖體激活受體γ共激活蛋白1 、慢速收縮肌
外文關鍵詞： Stra13, PGC-1α, P/CAF, MyoD, slow-twitch muscle fiber
相關次數： 點閱：15  下載：0 
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    • 成熟肌肉細胞一般分為兩種型態，第一型慢速收縮肌跟第二型快速收縮肌。在慢速收縮肌中，我們發現含有大量粒線體，因此可以藉由氧化電子傳遞鏈提供大量ATP提供人體生存所需的能量，反觀，快速收縮肌在能量代謝方面比較偏向醣解代謝，在外觀上，因為慢速收縮肌含有多血紅蛋白所以比較偏向紅色。目前我們知道PPAR-γ輔助轉錄因子PGC-1α在肌肉纖維的決定上有莫大關鍵性，而成體上主要表現在慢速收縮肌中。而之前文獻指出，轉殖鼠系統中，若過量表現PGC-1α在肌肉細胞中，會使得肌肉細胞走向慢速肌肉的趨勢，從之前觀察，我們知道在PGC-1α啟動子區有兩個非常保留E-box(CANNTG)，較轉錄起始點遠的E-box我們稱為E1-box，較轉錄起始點近的E-box稱為E2-box，由之前觀察我們知道MyoD 活化PGC-1α時，是藉由直接結合於PGC-1α啟動區的E2-box。藉由生物資訊分析另一個E-box是可能被Stra13所結合。從promoter assay，我們知道Stra13會抑制MyoD所主導PGC-1α的活化，而可能藉由的方式是經由跟MyoD競爭coactivator-P/CAF，而導致MyoD活化PGC-1α有所被抑制。

    Skeletal muscle are generally classified as two types – type I (slow - twitch) and type II (fast - twitch). The former is rich in mitchondria and thus provides constant ATP through oxidative metabolism. The latter depends on the glycolytic metabolism as the energy source. PGC-1α is a transcriptional coactivator mainly expressed in the slow-twitch fibers. Previous studies indicated that over-expression of PGC-1α promotes the conversion from fast-twitch fibers to slow-twitch fibers. According to previous observations, we know that the E2-box on the PGC-1α promoter is essential for MyoD-mediated transactivation. In this study, we found that Stra13, a putative E1-box binding transcriptional repressor, repressed the MyoD mediated PGC-1α promoter activation. The interaction between MyoD and Stra13 was almost undetectable by GST-Pull down assay and EMSA. In addition, over-expression of P/CAF, but not CBP, can rescue the Stra13-mediated repression. These data suggest that Stra13 represses MyoD-mediated PGC-1 activation by sequestering P/CAF from MyoD.

    聲明(Declaration) I 中文摘要 III Abstract IV 誌 謝 V 目 錄 VI 圖索引 X 表索引 XI 縮寫對照XII 第一章 緒論 1 1.1 肌纖維 1 1.2 PGC-1 ( peroxisome proliferative activated receptor, gamma, oactivator 1) family 3 1.2.1 PGC-1α 3 1.2.2 PGC-1β 5 1.2.3 PRC (peroxisome proliferative activated receptor, gamma, coactivator-related 1) 6 1.3 Stra13 7 1.4 研究動機與目的 8 第二章 材料與方法 10 2.1 細胞株 10 2.1.1 細胞培養 10 2.2 菌株 10 2.2.1 菌株培養 10 2.2.2 菌株保存 11 2.3 基本選殖技術 11 2.3.1 大腸桿菌勝任細胞之製備（Preparation of E. coli competent cells) 11 2.3.2 大腸桿菌的轉型作用 (Transformation) 11 2.3.3 質體DNA的少量製備 (Mini-preparation) – 用鹼處理法(Alkaline lysis method) 12 2.4 構築PGC-1 family promoter 12 2.4.1 Primer 設計 12 2.4.2 巢式聚合酶鏈反應 (nested Polymerase Chain Reaction, PCR) 13 2.4.3 PCR 條件 13 2.4.4 TA cloning 14 2.4.5 插入(Insert) DNA的純化 14 2.4.6 載體DNA的製備 15 2.4.7 載體DNA限制酶的剪切 16 2.4.8 載體DNA的5’端去磷酸根反應 16 2.4.9 載體DNA的純化 16 2.4.10 接合反應 ( Ligation ) 17 2.4.11 篩選 (Screening) 17 2.5 PGC-1 family promoter assay 17 2.5.1 Transfection 17 2.5.2 螢火蟲冷光活性方法 18 2.5.3 水母冷光活性方法 18 2.6 PGC-1α與蛋白質之交互作用 18 2.6.1 探針(probe)製備 18 2.6.2 探針的標定(labelling) 19 2.6.3 探針的純化 19 2.6.4 Binding assay 20 2.7 蛋白質純化 21 2.7.1 轉型作用 21 2.7.2 蛋白質表現及存化 21 2.7.3 蛋白質析出 21 2.8 蛋白質標定 22 2.9 GST-pull down assay 22 2.10 Competition assay 23 2.11 建立穩定表現Stra13及PGC-1αpromoter activity細胞株 23 2.11.1 細胞培養 23 2.11.2 轉染作用 23 2.11.3 挑single clony 24 2.11.4 構築pPYCAGIP-Stra13 24 2.11.5 Over-expression Stra13 C2C12建立 24 2.12 RT-PCR 25 2.12.1 Total RNA的抽取 25 2.12.2 反轉錄酶反應(Reverse Transcriptase, RT) 25 2.12.3 以PCR辨別PGC-1 family 在C2C12及Sol8表現 26 2.13 Real-time PCR 26 2.14 Western blotting 26 2.14.1 Stripping 27 第三章 結果 28 3.1 利用RT-PCR 觀察C2C12 及Sol8中PGC-1 family 的表現型態 28 3.2 觀察Stra13對於PGC-1 family的調控 29 3.3 Stra13對於MyoD 下游基因的調控方式 30 3.4 E-box對於MyoD活化PGC-1α是必要的，而Stra13是藉由E2 box達到抑制效果 30 3.5 探討MyoD及Stra13蛋白質和PGC-1α promoter 之間的作用關係 31 3.6 探討MyoD和Stra13之間交互作用關係 31 3.7 Stra13藉由與MyoD競爭coactivator達到抑制MyoD的活化倍率 32 3.8 建立overexpression-Stra13 PGC-1αpromoter stable clone 34 第四章 討論 36 第五章 結論 41 第六章 圖表 42 縮寫對照 62 參考文獻 63 Retrovirus製備的實驗流程圖 67 附錄一 68 附錄二 72 附錄三 73 藥品試劑 74 酵素和限制酶 74

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