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系統識別號 U0026-1708201014380000
論文名稱(中文) 研究hRad9蛋白在乳癌細胞中促進細胞老化與調控EMT上的功能性角色
論文名稱(英文) Study on the functional role of hRad9 protein in promoting senescence and modulating epithelial-mesenchymal transition in breast cancer
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 98
學期 2
出版年 99
研究生(中文) 王承凱
研究生(英文) Chen-Kai Wang
學號 s1697107
學位類別 碩士
語文別 中文
論文頁數 102頁
口試委員 指導教授-張敏政
口試委員-張明熙
口試委員-賴明德
口試委員-鄭宏祺
中文關鍵字 細胞老化  hRad9  上皮-間質轉化 
英文關鍵字 senescence  hRad9  EMT 
學科別分類
中文摘要 Rad9 蛋白質最早是在Schizosaccharomyces prombe 被發現,而進一步在人類身上也同樣發現Rad9的同源蛋白,hRad9,hRad9是checkpoint rad gene的其中一員,是個具有高度保存性的蛋白並且hRad9在細胞中參與了許多不同重要的生理功能。雖然有許多研究文獻報導hRad9蛋白的各種生理功能但是hRad9蛋白在人類細胞癌症中所扮演的真正角色並未明確。細胞老化被認為是細胞保護自己的反應機制之ㄧ並且會出現在較良性的腫瘤中。在之前的相關報導中,在乳癌肺癌中觀察到hRad9蛋白有累積的情形,並且hRad9蛋白被認為是個腫瘤致癌基因;然而,一些研究顯示hRad9蛋白是腫瘤抑制蛋白因為hRad9保持基因的完整度和抑制androgen receptor (AR)的活性。而為了釐清hRad9蛋白在癌症中扮演的角色所以先在MDA-MB-231 breast cancer cell lines 中建立hRad9蛋白大量表現穩定的細胞株(此細胞株已由實驗室凡志學長建立),下一步我們對於細胞型態、細胞生長速度、細胞爬行能力以及形成聚落的能力進行觀察。結果發現在hRad9大量表現的細胞株中,在細胞型態上可以發現有部分細胞呈現較肥大並有較攤平的外觀並且在細胞生長速率、細胞爬行速度及形成聚落的能力,皆有下降的趨勢。由於細胞的外觀改變並有生長速率的降低的特色和已報導的現象細胞老化( Senescence )相符,故利用其他老化相關特色,例如:Senescence associated β-galactosidase、Stress fibers actin 經由觀察後更進一步發現這類型的細胞有表現老化細胞的特徵出現,藉由這些特徵顯示大量表現hRad9蛋白會使細胞呈現老化的現象;另一方面為了更確定hRad9蛋白的角色定位,我們所取得的invasive ductal carcinoma specimens中有發現大部分檢體呈現hRad9 蛋白低量表現的情形,並且在良性與惡性不同的細胞株中,觀察到較惡性並較具侵犯性的細胞株,hRad9蛋白呈現低表現,這給了我們暗示hRad9在腫瘤惡化程度中可能扮演著重要的角色,因此我們認為hRad9蛋白的下降似乎和細胞較惡化具有一定關係存在,故我們以核酸干擾技術暫時抑制MCF-7 breast cancer cell lines hRad9的表現,從細胞外型中發現在細胞型態上產生改變並且在細胞的爬行能力有上升的表現。並且發現細胞的爬行速率也有上升的情形,進而在西方墨點法技術中,發現Epithelial marker like E-cadherin的減少和Mesenchymal markers like Vimentin的增加,然而此EMT marker的改變是否真的造成在動物model中細胞株轉移能力的增加,還需要進一步的研究探討。綜合以上結果,我們認為hRad9可能透過調控下游因子及EMT相關蛋白進而在乳癌細胞中扮演一個抑癌的角色。而更詳細的機制還必須在未來做更詳盡的探討。
英文摘要 The hRad9 protein is a structural homologue to Schizosaccharomyces pombe rad9 in human beings, which is a member of the Rad family of checkpoints that plays important roles in several fundamental biological processes. Cellular senescence is an anti-proliferative response considered as a tumor suppressive mechanism. Accumulation of hRad9 has been reported in breast, lung and prostate cancers, and suggest that hRad9 may act as an oncogenic protein. However, several studies also support that hRad9 might serve as a tumor suppressor due to its functions in maintaining chromosome integrity and repressing androgen receptor (AR) activity. In this study, We established stably hRad9-overexpressing cells in MDA-MB-231, 231-CR9, and determined their tumorigencity. In comparison with the mock-transfected cells, 231-PC, the morphology of 231-CR9 cell was enlarged and vacuolous. The cell growth rate of 231-CR9 was lower than that of 231-PC cells. In addition, migration abiliyty of 231-CR9 was decreased when compared to that of 231-PC cells. Several features of senescent cells including large size cell , the enrichment stress fibers , and the senescence-associated β-galactosidase (SA-β-gal) was observed in 231-CR9 , and further we knock-down hRad9 expression resulted in the reversal of cellular senescence in 231-CR9, to suggest that hRad9 was associated with cellular senescence in 231-CR9. Knock-down of hRad9 resulted in loss of cell-cell adhesion epithelial markers like E-cadherin , activation of mesenchymal markers, and induction of cell motility. To suggest that reduced hRad9 protein might contribute to metastasis by promoting an epithelial-mesenchymal transition (EMT). Put together, our data indicated that hRad9 protein was involved in regulating senescence and epithelial-mesenchymal transition in cancer cells. We will investigate the mechanisms of hRad9-mediated senescence EMT as well as confirm our hypothesis that hRad9 protein play an important role in promoting senescence and modulating epithelial-mesenchymal transition in vivo in the future.
論文目次 目錄
中文摘要 I
Abstract III
致謝 V
目錄 VII
圖表目錄 X
縮寫表 XII
第1章緒論 1
1-1 hRad9蛋白簡介 1
1-2 hRad9蛋白功能性區域 3
1-3 hRad9蛋白的生理功能 4
1-3-1 DNA repair 4
1-3-2 Cell cycle 5
1-3-3 Apoptosis 7
1-3-4 Transcription activity 8
1-3-5 Genomic integrity 9
1-3-6 Cancer 9
1-4細胞老化(Senescence) 10
1-4-1細胞老化的發現 11
1-4-2造成細胞老化的因素 12
1-5細胞週期(cell cycle) 16
1-5-1細胞週期的定義 17
1-6細胞老化之分子機制(molecular mechanism of cellular senescence) 18
1-6-1 p53-dependent路徑 18
1-6-2 p53-independent路徑 20
1-7研究動機 21
第2 章材料與方法 22
2-1實驗菌株、載體與培養基配方 22
2-1-1實驗菌株 22
2-1-2載體 (附錄2 ) 22
2-1-3培養基配方 22
2-2細胞培養方法 23
2-2-1實驗細胞株 23
2-2-2細胞解凍 24
2-2-3細胞繼代培養 (附著型細胞 adherent cell) 25
2-2-4細胞計數 26
2-2-5細胞保存 27
2-3質體製備 28
2-3-1聚合酶連鎖反應 28
2-3-2構築 PCR 片段於質體中 29
2-3-3 E. coli 形質轉移 30
2-3-4小量質體製備 31
2-4蛋白質分析 32
2-4-1蛋白質萃取 32
2-4-2蛋白質定量 33
2-4-3 SDS-PAGE 蛋白質電泳 34
2-4-4西方墨點法 35
2-5在 MCF-7 前期乳癌細胞建立 hRad9 表現抑制的穩定細胞 37
2-5-1細胞轉染 37
2-5-2利用抗生素篩選穩定細胞株 38
2-6大量表現的穩定細胞株對於影響腫瘤生長能力的分析 39
2-6-1利用螢光染色觀察穩定細胞株之肌動蛋白及應力纖維之活動情形 39
2-6-2利用Cell counting 試驗偵測穩定細胞株之生長能力 40
2-6-3利用傷痕癒合試驗( Wound healing )分析穩定細胞株之爬行能力 40
2-6-4利用Boyden chamber分析穩定細胞株之爬行能力 41
2-6-5利用Ki-67分析穩定細胞株之proliferation 42
2-6-6利用Propidium Iodide (PI)-AnnexinⅤ雙染實驗分析穩定細胞株之細胞凋亡 43
2-6-7利用Senescence detection Kit實驗分析細胞株之細胞老化 44
2-7統計方法 45
第3章實驗結果 46
3-1在 MDA-MB-231 後期乳癌細胞建立 hRad9 大量表現的穩定細胞株並且觀察其穩定細胞株的型態。 46
3-2 hRad9 大量表現的穩定細胞株對於影響腫瘤生長能力的分析 47
3-2-1利用相位差顯微鏡觀察hRad9 蛋白大量表現時對於細胞型態上的影響 47
3-2-2利用Cell counting assay 試驗和 Colony formation assay偵測穩定細胞株之生長能力 48
3-2-3利用Ki-67 proliferation Marker 探討hRad9大量表現穩定細胞株對生長能力的影響 49
3-2-4利用Annexin Ⅴ和Propldium Iodide ( PI)染劑 探討hRad9大量表現穩定細胞株對細胞凋亡的影響 50
3-2-5利用Rhodamine phalloidin螢光染色觀察穩定細胞株之肌動蛋白及應力纖維之活動情形 51
3-2-6利用Senescence associated β–galactosidase染色觀察穩定細胞株的 變化 52
3-2-7利用傷痕癒合試驗分析穩定細胞株之爬行能力 53
3-2-8利用 Boyden chamber分析穩定細胞株之爬行能力 54
3-2-9觀察穩定細胞株之產生細胞老化的機轉 55
3-3在 MCF-7 前期乳癌細胞暫時轉染 siRad9 表現抑制的細胞株 56
3-4 hRad9 表現抑制的暫時轉染細胞株對於影響腫瘤生長能力的分析 57
3-4-1利用相位差顯微鏡觀察hRad9 蛋白表現受抑制時對於細胞型態上的影響 57
3-4-2利用 Boyden chamber分析暫時轉染細胞株之爬行能力 58
3-4-3觀察暫時轉染細胞株之EMT ( Epithelial-mesenchymal transition ) marker表現的情形 58
3-5在 MCF-7 前期乳癌細胞建立 hRad9 表現抑制的穩定細胞株 60
3-6 hRad9 表現抑制的穩定細胞株對於影響腫瘤生長能力的分析 61
3-6-1利用相位差顯微鏡觀察hRad9 蛋白表現受抑制時對於細胞型態上的影響 61
3-6-2利用 Boyden chamber分析穩定細胞株之爬行能力 62
3-6-3觀察穩定細胞株之EMT ( Epithelial-mesenchymal transition ) marker表現的情形 62
3-6-4利用螢光染色( Immunofluorescence )觀察穩定細胞株之EMT ( Epithelial-mesenchymal transition ) marker情形 64
3-6-5利用西方墨點法( Western blot )觀察暫時轉染細胞株細胞株中EMT ( Epithelial-mesenchymal transition )相關轉錄因子表現 64
第4章結論 66
第5章實驗討論 68
5-1 hRad9 蛋白在癌症中所扮演的角色 68
5-2過度表現hRad9 蛋白在乳癌細胞中所扮演的角色 69
5-3抑制hRad9 蛋白對於乳癌細胞致癌性的影響 71
5-4 hRad9 蛋白在癌症中扮演著雙向的功能性角色 72
實驗結果圖表 75
附錄1 、Human Rad9 shRNA sequence 94
附錄2、實驗所用載體各區域圖 95
參考文獻 97
自述 102

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