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系統識別號 U0026-2008201315090500
論文名稱(中文) 碳水化合物反應元件結合蛋白轉錄因子在細胞壓力反應下的研究
論文名稱(英文) The biological significance of carbohydrate responsive element binding protein in response to cellular stress
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 101
學期 2
出版年 102
研究生(中文) 丁雅柔
研究生(英文) Ya-Rou Ding
學號 T16994036
學位類別 碩士
語文別 英文
論文頁數 52頁
口試委員 指導教授-周楠華
口試委員-蔣輯武
口試委員-劉校生
中文關鍵字 癌症  壓力因素  碳水化合物反應元件結合蛋白轉錄因子 
英文關鍵字 cancer  biological stresses  carbohydrate responsive element binding protein 
學科別分類
中文摘要 在癌症發展過程中,有許多壓力因素會造成癌細胞惡化,其中最重要的是癌症胞對抗缺氧壓力以及逃避細胞凋亡的機制。在我們先前的研究中證明: 有受體酪氨酸激酶---RON大量表現的膀胱癌細胞,在缺乏血清的環境下,會扮演轉錄因子的角色,活化一系列的基因表現,促成癌細胞對於環境中壓力的調整以及適應。碳水化合物反應元件結合蛋白轉錄因子(ChREBP)是bHLH/LZ 轉錄因子家族中的一員。ChREBP本身為轉錄因子,並且可調控許多基因。之前的研究說明,ChREBP可以調控脂質合成以及糖解作用相關的基因。我們之前的研究發現,ChREBP在去除血清的壓力情況下,很可能是膀胱癌細胞誘發出來的壓力反應相關基因之一。本研證明, ChREBP廣泛表現於人類泌尿上皮細胞、膀胱癌細胞以及肝癌細胞。而隨著肝癌細胞惡化的程度,ChREBP表現量會相對減低。當葡萄糖完全去除的情況下,ChREBP的位置依舊維持在細胞質,而不會進入細胞核。然而,在恢復環境中的高葡萄糖濃度時,ChREBP的會進入細胞核進行它應有的功能以及調控。在完全去除血清的壓力情況下,缺氧的環境下,以及生長因子刺激下, ChREBP在癌細胞的位置會進入細胞核。我們進一步也證明,在膀胱癌細胞大量表現ChREBP的情況下,會促進細胞增生、細胞爬行的能力以及細胞的非貼附性生長。因此我們可以得到一個結論,ChREBP可能在癌症發展過程中扮演一個正向的角色,未來可從這方面著手研究成為癌症治療的標靶。
英文摘要 Human body is exposed to different biological stresses from external and internal environments. Normal cellular responses to stress are important barriers to carcinogenesis. These cellular stresses and evasion of apoptosis are important biological characteristics of tumorigenesis. Our recent study demonstrated that over-expression of receptor tyrosine kinase-RON may provide human cancer cells with stress adaptation advantage by functioning as a transcriptional activator. The carbohydrate responsive element binding protein (ChREBP) belongs to bHLH/LZ transcription factor family, and was reported to regulate several genes related to lipogenesis and glycolysis. ChREBP was one of the candidate target genes for nuclear RON in response to serum starvation in human bladder cancer. We demonstrated that ChREBP is ubiquitously expressed in human uroepithelial cells and liver cancer cell lines, with a trend toward down-regulation in the progression of hepatocarcinogenesis. Most of ChREBP was localized to the cytosol in the absence of glucose, and translocated into nucleus after glucose supplement. In contrast, ChREBP was translocated into nucleus in response to serum starvation, hypoxic stress and growth factor stimulation onto cancer cells. Taken together, ChREBP not only functions as a glucose-sensing protein but also a stress-response protein. Overexpression and/or activation of ChREBP promote the proliferation, migration and anchorage-independent growth. ChREBP may play a positive role in the cancer progression and thus deserves to be investigated as a therapeutic target in the treatment of cancer in the future.
論文目次 Introduction...............................................1
Cellular stress in the cancer..............................1
Hypoxia ...................................................2
Serum starvation...........................................3
Glucose deprivation........................................4
Warburg effect.............................................4
Carbohydrate response element binding protein (ChREBP).....5
Material and method........................................8
Cell lines.................................................8
Western blotting...........................................8
Immunofluorescent microscopy and confocal imaging analysis ...........................................................9
Hypoxic challenge..........................................9
Serum starvation..........................................10
Glucose depletion and restoration experiments.............10
EGF treatment.............................................10
Construction of ChREBP plasmid............................11
Transient transfection....................................12
Stable clone establishment................................12
Cell proliferation assay..................................13
Wound healing assay.......................................13
Soft agar assay...........................................14
RESULTS...................................................15
Expression of ChREBP in bladder cancer cell line and hepatoma cell line pattern................................15
Expression ChREBP response to serum-starvation in TSGH8301 cell line.................................................15
Expression of ChREBP in response to glucose deprivation and restoration in TSGH8301 cell line.........................16
Expression of ChREBP in low glucose and hypoxia stress in TSGH8301 cells............................................17
Expression of ChREBP in response to hypoxia in TSGH8301 cells.....................................................17
Expression of ChREBP in response to EGF stimulation in TSGH8301 cell line........................................18
Expression of ChREBP in response to hypoxia in HepG2 cell line......................................................19
Interaction of with phosphatase 2A (PP2A) subunits in HeLa cells.....................................................19
The biological functions ChREBP of 293-ChREBP stable clones ..........................................................20
Discussion................................................22
Reference.................................................25
Figure....................................................32


Content of Figures
Figure 1. The Express pattern of ChREBP in uroepithelial cell lines................................................33
Figure 2. The Express pattern of ChREBP in human hepatoma cell lines................................................34
Figure 3. The subcellular distribution of ChREBP expression in TSGH8301 cancer cells under serum starvation...........35
Figure 4. The subcellular distribution of ChREBP expression in TSGH8301 cancer cells under glucose derivation.........36
Figure 5. Time course expression of ChREBP in TSGH8301 cancer cells in response to glucose depletion.............37
Figure 6. The subcellular distribution of ChREBP expression in TSGH8301 cancer cells in response to glucose restoration. ..........................................................38
Figure 7. Time course expression of ChREBP in TSGH8301 cancer cells in response to glucose restoration...........39
Figure 8. The subcellular distribution of ChREBP expression in TSGH8301 cancer cells in response to hypoxia and low-glucose stress............................................40
Figure 9. Time course expression of ChREBP and HIF-1a in TSGH8301 cancer cells in response to low-glucose and hypoxic stress....................................................41
Figure 10. The subcellular distribution of ChREBP expression in TSGH8301 cancer cells in response to hypoxia...........42
Figure 11. Time course expression of ChREBP and HIF-1a in TSGH8301 cancer cells cultured in the high-glucose medium (25mM)....................................................43
Figure 12. The subcellular distribution of ChREBP expression in TSGH8301 cancer cells after EGF treatment..............44
Figure 13. The subcellular distribution of ChREBP expression in Hep G2 cells in response to hypoxia....................45
Figure 14. Time course expression of ChREBP and HIF-1a in HepG2 cells in response to hypoxia........................46
Figure 15. The subcellular distribution of ChREBP in relation to PP2A subunit in HeLa cells....................47
Figure 16. The establishment of 3x-FLAG-ChREBP-myc construct.................................................48
Figure 17. The effect of ChREBP on cell growth in vitro...50
Figure 18. The effect of ChREBP on cell migration in vitro.....................................................51
Figure 19. The significance of ChREBP on cell colony formation.................................................52
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