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系統識別號 U0026-0812200914285025
論文名稱(中文) 探討肝脂合成作用中C/EBPD參與在HDAC1/HDAC3調控PPARG2基因轉錄作用之機轉
論文名稱(英文) HDAC1/HDAC3 modulates PPARG2 transcription through sumoylated C/EBPD in hepatic lipogenesis
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 96
學期 2
出版年 97
研究生(中文) 賴姵樺
研究生(英文) Pei-Hua Lai
學號 s2695411
學位類別 碩士
語文別 中文
論文頁數 95頁
口試委員 指導教授-張文昌
指導教授-王育民
口試委員-蔡曜聲
中文關鍵字 PPARG2  CEBPD  Lipogenesis  HDAC1/3  Sumoylation 
英文關鍵字 PPARG2  CEBPD  Lipogenesis  HDAC1/3  Sumoylation 
學科別分類
中文摘要 C/EBPD與PPARG2在調控脂質代謝基因上扮演重要角色。本實驗室研究成果顯示在人類肝癌細胞(HepG2)中,過度表現的C/EBPD可促進脂肪堆積現象並活化PPARG2轉錄作用。將PPARG2基因啟動區的5’端進行段切並分析報導基因表現,發現啟動區中-457/-129bp區域C/EBPD調控區段。以凝膠位移電泳及點突變實驗得知C/EBPD結合至此區段上的-324/-311及-158/-145bp兩個位置進而調控PPARG2基因啟動區的活化。本實驗室先前研究發現C/EBPD可被SUMO1進行sumoyaltion的後轉譯修飾作用,我們進一步也在HepG2細胞中證實C/EBPD的第120個胺基酸-lysine位置可被sumoylation (suC/EBPD),進而召集HDAC1及HDAC3。同時,利用ChIP assay得知無法被sumoylation的mutant即喪失和HDAC1及HDAC3的交互作用。在lipogenic stimulator處理下,C/EBPD的sumoylation程度不變,卻大量誘導出C/EBPD蛋白質;意味著過量且未被sumoylation的C/EBPD可能扮演關鍵性的活化角色,進而逆轉原本suC/EBPD/HDAC1/HDAC3抑制性複合體對PPARG2基因的不活化效應並進一步促進肝癌細胞的脂質生成作用。綜觀以上研究,我們證實了suC/EBPD/HDAC1/HDAC3抑制性複合體能夠抑制PPARG2基因的轉錄作用,而大量表現的C/EBPD能夠短暫性地活化PPARG2基因表現並且參與HepG2細胞的脂質生成作用。
英文摘要 CCAAT/Enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors gamma (PPARG) play critical roles in the regulation of lipid metabolism genes. Overexpression of C/EBPDelta (C/EBPD) enhances lipid accumulation and specifically activates PPARG2 transcription in HepG2 cells. By using 5'-serial deletion reporter analysis, we show that the region comprising the -457 to +129 base pairs is required for C/EBPD response of the PPARG2 promoter. Two critical C/EBPD-binding motifs on the -324/-311 and -158/-145 of human PPARG2 promoter are identified. We previously have shown that the human C/EBPD is sumoylated by small ubiquitin-related modifier-1 (SUMO1). We further demonstrated the sumoylation of C/EBPD lysine 120 is also detectable in HepG2 cells, and this modification functions for binding of the recruits, HDAC1 and HDAC3. Meanwhile, an in vivo chromatin IP assay demonstrated that the sumoylation mutant of C/EBPD lost a significant portion of HDAC1 and HDAC3 interaction. Combining that the increasing amount of C/EBPD and the sumoylated C/EBPD (suC/EBPD) consistently responded to lipogenic stimulation, these results suggest that the excess non-sumoylated C/EBPD could be a critical activator which reverses suC/EBPD/HDAC1/HDAC3-mediated PPARG2 gene inactivation and promotes hepatic lipogenesis. Taken together, we suggest that suC/EBPD/HDAC1/HDAC3 complex inactivates PPARG2 transcription, and the induction of C/EBPD expression transiently activates PPARG2 transcription are involved in adipocyte-like lipogenesis in HepG2 cells.
論文目次 中文摘要..................................................1
Abstract..................................................3
縮寫檢索表................................................5
第一章 緒論...............................................6
1. 脂肪細胞分化作用 (Adipogenesis)........................6
2. 脂肪生成作用(Lipogenesis)..............................9
3. CCAAT/enhancer-binding proteins.......................10
4. Peroxisome proliferator-activated receptors γ,PPARG..11
5. 染色質重組酵素與脂肪前驅細胞分化作用的關係............12
6. 研究動機..............................................13
7. 研究目標..............................................14
第二章 實驗材料..........................................15
第三章 實驗方法..........................................20
1. 細胞培養..............................................20
2. Oil red O中性脂肪染色與定量...........................20
3. 細胞株之脂肪堆積作用..................................21
4. 細胞全量RNA萃取.......................................28
5. 反轉錄反應(reverse transcription, RT).................29
6. PCR...................................................30
7. 西方點墨法(Western blotting)..........................31
8. 測定人類PPARG2基因啟動區的promoter activity...........33
9. 分析PPARG2 promoter DNA序列與核蛋白質結合狀況.........35
第三章 實驗結果..........................................39
1. C/EBPD參與肝癌細胞肝脂合成作用........................39
2. C/EBPD活化PPARG2基因表現及啟動區活性..................41
3. C/EBPD結合至PPARG2啟動區上的兩個C/EBPD結合位置並調控其轉錄活性...................................................43
4. C/EBPD可於肝癌細胞(HepG2 cells)中進行 sumoylation.....44
5. suC/EBPD在HDAC1及HDAC3抑制PPARG2基因啟動區活性的效應上扮演關鍵性角色.............................................49
6. suC/EBPD、HDAC1及HDAC3三者在PPARG2基因啟動區上形成蛋白複合體調控其轉錄作用 .......................................52
第四章 討論..............................................55
第五章 未來展望與研究....................................64
第六章 參考文獻..........................................65
附圖.....................................................74
附錄.....................................................93
自述.....................................................95
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