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系統識別號 U0026-0812200915205988
論文名稱(中文) hRad9 蛋白在人類癌症細胞株中的功能性探討
論文名稱(英文) Functional studies of hRad9 in human cancer cell lines
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 97
學期 2
出版年 98
研究生(中文) 王惠柔
研究生(英文) Hui-Jou Wang
學號 s1696408
學位類別 碩士
語文別 中文
論文頁數 75頁
口試委員 口試委員-鄭宏祺
口試委員-賴明德
指導教授-張敏政
口試委員-張明熙
中文關鍵字 A549肺癌細胞  hRad9蛋白  細胞凋亡  EMT 
英文關鍵字 epithelial-mesenchymal transition  hRad9  apoptosis  A549 
學科別分類
中文摘要 人類的Rad9蛋白為Schizosaccharomyces pombe Rad9蛋白的同源蛋白,其為checkpoint rad genes產物中的一員,是一個在演化上具有高度保留性的蛋白。hRad9在細胞內扮演著許多重要的角色,包括偵測DNA的受損、調節細胞週期的進行 、執行 DNA 的修復等等。雖然目前已有許多文獻報導hRad9所具有的生理功能,但對於它在癌症裏頭所具有的生理意義始終沒有一個確切的結論,而為了釐清hRad9在腫瘤形成與惡化過程中所扮演的角色,我們利用人類癌症細胞株來對hRad9作功能性探討。首先我們先在A549這株肺癌細胞裡頭建立hRad9表現抑制的穩定細胞株,接著我們對細胞型態(cell morphology)、細胞生長速率(proliferation)、細胞爬行能力(migration)以及形成聚集之能力(anchorage-independent growth abilities)進行觀察,發現在hRad9表現抑制穩定細胞株裡頭,不論是細胞生長速率、或是形成聚集之能力,相較於送入空載體的對照組,都有上升的現象。此外細胞爬行能力也有上升的情形,而我們也更進一步發現與EMT有關的蛋白--- N-cadherin和vimentin的表現量是上升的。同時我們以核醣核酸干擾技術抑制MCF7前期乳癌細胞內hRad9蛋白的表現,則發現E-cadherin的蛋白表現量是下降的。另外我們在A549肺癌細胞裡也同時建立了hRad9過度表現穩定細胞株,做了上述特性的分析後,發現細胞生長速率、細胞爬行能力以及形成聚集之能力下降,而N-cadherin和vimentin的表現量也是下降的。接著我們利用西方點墨法更發現hRad9表現抑制穩定細胞株裏頭,Bcl-2這個抗細胞凋亡的蛋白,表現量有上升的情形,因此我們認為細胞形成聚集之能力上升可能有部分源自於Bcl-2的過度表現,而造成細胞抗凋亡的能力上升所致。另一方面由MTT測定法的結果也發現,hRad9表現抑制穩定細胞株對於camptothecin(CPT)這個會造成DNA損傷的藥物有比較高的耐受性,綜合以上結果,我們認為hRad9可能透過部分調控EMT相關蛋白及Bcl-2而在A549細胞裡頭扮演一個抑癌的角色。而其中詳細調控的機制還有待未來做更詳盡的探討。
英文摘要 The human Rad9, a homologue of yeast Schizosaccharomyces pombe rad9, is a member of the Rad family of checkpoint proteins. It is involved in detection of DNA damage, cell cycle arrest, and DNA repair. Although considerable information about hRad9 and its function has been accumulated, its behavior in malignant cells has not been examined. To further investigate the biological functions of hRad9 in human cancer cell lines, we established hRad9 knocked down stable cell lines in A549 cells(hRad9-shRNA cells)and then cell morphology, proliferation, migration and anchorage-independent growth abilities of hRad9-shRNA cells were examined. The growth rate of hRad9-shRNA cells in DMEM medium was higher than that of its empty vector-transfected cells. hRad9-shRNA cells also exhibited a statistically significant increase in the number of colonies compared with its empty vector-transfected cells, suggesting that hRad9 knocked down in A549 cells resulted in increasing its anchorage-independent growth potential. In addition, the results of cellular migration assay also revealed an increase in migration ability of hRad9-shRNA cells when compared to that of its empty vector- transfected cells. Furthermore, western blot analysis also revealed that the protein levels of EMT hallmarks, N-cadherin and vimentin, were upregulated in hRad9-shRNA cells. On the other hand, we found that growth rate, colony formation and migration abilities decreased as well as EMT hallmark, N-Cadherin and Vimentin, were downregulated in hRad9- overexpressing stable cell lines. Furthermore, we also found that the protein level of Bcl-2 significantly increased in hRad9-shRNA cells, suggesting the enhancement of colony formation ability observed in hRad9-shRNA cells likely resulted from the overexpression of Bcl-2 which promotes the anti-apoptotic ability. In addition, the results of MTT assay revealed that hRad9-shRNA cells exhibited more resistant to camptothecin than its empty vector-transfected cells did when the cells were exposed to camptothecin. Put together, our preliminary data indicated that hRad9 is a tumor suppressor in A549 cells and its tumor suppression is mediated at least in part through the regulations of EMT hallmarks and Bcl-2. The detailed mechanism of hRad9-mediated tumor supression in A549 cells or other cancer cells will be investigated in the near future.
論文目次 中文摘要………………………………………………………I
英文摘要………………………………………………………III
誌謝……………………………………………………………V
目錄……………………………………………………………VI
縮寫檢索表……………………………………………………X
緒論………………………………………………………………1
一、hRad9蛋白……………………………………………………1
1、hRad9蛋白分子量 ( Mr )………………………………2
2、hRad9蛋白的功能性區域 ( functional domain )……2
3、hRad9蛋白主要的生理功能 ( biological functions )…3
(a) hRad9與DNA修復( DNA repair )的調控機制……3
(b) hRad9與細胞週期( cell cycle )的調節機制……5
(c) hRad9與基因體完整性( genomic integrity )之影響…6
(d) hRad9與細胞凋亡( apoptosis )的調控關係……6
(e) hRad9與癌症的關係………………………………7
研究動機…………………………………………………………9
材料與方法……………………………………………………11
ㄧ、使用之菌株、載體及培養基………………………………11
二、少量質體 DNA 的抽取……………………………………12
三、DNA 濃度測定……………………………………………13
四、hRad9基因全長與p3xflag載體之建構……………………14
五、接合反應 ( Ligation )………………………………………15
六、質體DNA在大腸桿菌的轉形作用(Transformation)……16
七、細胞培養……………………………………………………17
八、細胞轉染(transfection)………………………………………19
九、利用抗生素篩選穩定細胞株………………………………20
十、收取細胞 (Harvesting cell lysates)………………………21
十一、蛋白質濃度的定量………………………………………22
十二、SDS-PAGE之蛋白質分子量分析……………………………23
十三、西方墨點法 (Western blotting)…………………………25
十四、利用MTT assay偵測穩定細胞株之生長速率…………27
十五、利用 Boyden chamber分析穩定細胞株之爬行能力…28
十六、軟洋菜膠細胞群落形成法(Soft agar colony formation
assay)……………………………………………………30
十七、DNA損傷藥物CPT( camptothecin )處理………………31
實驗結果 …………………………………………………………33
一、在A549肺癌細胞中建立hRad9表現抑制穩定細胞株並觀察其細胞
外觀的變化…………………………………33
二、觀察在hRad9表現抑制穩定細胞株裡,與EMT相關蛋白之變化.34三、觀察hRad9表現抑制穩定細胞株的爬行能力……………35
四、利用核醣核酸干擾技術以及暫時轉染hRad9觀察其對EMT相關蛋
白之影響……………………………………35
五、觀察hRad9表現抑制穩定細胞株的生長速率……………36
六、觀察hRad9表現抑制穩定細胞株的非固著依賴性生長能力……36
七、探討hRad9可能影響細胞形成聚集能力之機制…………37
八、在細胞處理DNA損傷藥物(CPT)之後,hRad9對於細胞凋亡之影
響…………………………………………………………………38
九、在A549肺癌細胞中建立hRad9過度表現穩定細胞株並觀察其細胞
外觀的變化…………………………………39
十、觀察在hRad9過度表現穩定細胞株裡,與EMT相關蛋白之變化.40
十一、觀察hRad9過度表現穩定細胞株的爬行能力…………41
十二、觀察hRad9過度表現穩定細胞株的生長速率…………41
十三、觀察hRad9過度表現穩定細胞株的非固著依賴性生長能力…42
十四、在細胞處理DNA損傷藥物(CPT)之後,過度表現hRad9對於細
胞凋亡之影響…………………………42
實驗討論………………………………………………………43
結論………………………………………………………………48
實驗圖表……………………………………………………………49
附錄……………………………………………………………………66
參考文獻……………………………………………… 69
自述……………………………………………………………75
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