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研究生:馮文萱
研究生(外文):FENG Wen-Hsuan
論文名稱:探討RibosomalproteinS19在大腸直腸癌細胞株所扮演的角色
論文名稱(外文):The study of Ribosomal protein S19 roles in colorectal cancer cell lines
指導教授:黃世明黃世明引用關係黃紀榕黃紀榕引用關係
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:98
語文別:中文
論文頁數:49
中文關鍵詞:大腸癌抗癌藥物核醣體蛋白質核醣體蛋白質小次單位19
外文關鍵詞:RPS19Ribosomal proteinColon cancerOxaliplatin
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核醣體蛋白質小次單位19(Ribosomal protein S19, RPS19)是構成核醣體小次單位40S的組成蛋白質,先前研究發現核醣體蛋白質除了擔任核醣體的形成,也具備調控基因轉錄的功能。本實驗室使用大腸癌患者的糞便檢體抽取RNA後,經反轉錄成cDNA使用生物晶片偵測發現在大腸癌前期病人中核醣體蛋白質小次單位19基因大量表現,本研究將探討RPS19在大腸癌前期所參與基因轉錄調控的對象及機制。論文中使用大腸癌細胞株Hct116 p53+/+與Hct116 p53-/-,利用反轉錄病毒建立降解RPS19 mRNA的穩定細胞株(sh-RPS19),將控制組細胞株(sh-Luc)與實驗組細胞株(sh-RPS19)分別以MTT和流式細胞儀分析細胞生長以及週期的差異性,以及使用Real Time PCR及西方墨點法觀察在抗癌藥物Oxaliplatin作用下特定基因和蛋白質的表現量。由實驗結果發現細胞在核醣體蛋白質小次單位19基因降低的情況下,無論p53有無皆會使細胞生長較為緩慢且細胞週期在G0/G1期有停滯的現象;在作用藥物Oxaliplatin的實驗中,發現sh-RPS19細胞株在細胞週期和蛋白質表現上發現sub-G1增加以及p21、r-H2AX蛋白質有大量表現的情形,推測似乎對於藥物反應具有加乘反應的效果以及參與細胞凋亡或修補機制;論文中也探討了sh-RPS19細胞株可能透過抑制26S proteasome路徑來達到穩定p21蛋白質的表現。由實驗結果推論細胞缺乏RPS19基因表現會造成細胞生長受到抑制,並且對於藥物作用可能有加強的反應效果。最近幾年有關核醣體蛋白質小次單位19的結構以及相關作用蛋白質陸續發現,也有文獻指出核醣體蛋白質小次單位19可能參與了細胞中轉錄的機制,未來有關核醣體蛋白質小次單位19在癌細胞扮演的角色還需更進一步的探討。
Ribosomal protein S19 is a component of 40S Ribosome subunit,previous study found Ribosomal protein S19 except to participate in ribosome synthesis, also have the functions of gene translation regulations. In this study, we used Biochip assay screening cDNA from colon cancer patient stool which detected the RPS19 gene overexpressed in primary human colon cancer patients. To understand the rule of RPS19, Hct116 p53+/+ and Hct116 p53-/- cells infected with lentivirus expressing RPS19 siRNA to established sh-Luc and sh-RPS19 cell lines. The effects of RPS19 on colon cancer cell growth and cell cycle were evaluated with MTT assay and Flow cytometry, the data showed decreased RPS19 gene caused cells grew much slower and induced G0/G1 arrest, we also treated Oxaliplatin, a chemotherapeutic drug, caused p21 and r-H2AX induction in sh-RPS19 cell line, and furthermore we speculated that decreased RPS19 expression may blocks 26S proteasome pathway than accumulated p21 protein. In conclusion, the study suggested decrease RPS19 gene expression may inhibited cancer cell growth and exhibited synergistic effect on Oxaliplatin treatment. In recent years there has been found many research of RPS19 protein structures and interaction proteins, they also indicated that RPS19 may be an important role in translation regulation. The molecular mechanisms of RPS19 in the tumorigenesis still have more study to investegated.
目錄
目錄 Ⅰ
圖目錄 Ⅱ
縮寫表 Ⅲ
中文摘要 Ⅳ
英文摘要 Ⅴ
第一章 緒論 9
第二章 實驗材料與方法 13
第一節 實驗材料 13
第二節 實驗方法 17
第三章 結果 27
第一節 利用慢病毒(lentiviral)系統降低大腸癌細胞
HCT116細胞RPS19 mRNA表現量

27
第二節 分析有無p53表現細胞株在RPS19表現量下降的情況對於細胞生長及細胞週期的影響

27
第三節 觀察Oxaliplatin對於HCT116 p53+/+ sh-Luc和sh-RPS19細胞株的影響

28
第四節 RPS19對於p21蛋白質的穩定性 30
第四章 討論 31
第一節 RPS19可能參與癌細胞生長 31
第二節 降低RPS19表現量可能會加乘Oxaliplatin的作用

32
第三節 RPS19可能主要參與在轉錄後調控 32
第四節 降低RPS19可使細胞藉由-H2AX造成細胞凋亡或修補

33
第五節 降低RPS19可能由阻斷proteasome 使p21蛋白質更加穩定

34
第六節 sh-RPS19在mRNA 與蛋白質效力的差異性 34
圖 37
參考文獻 46

圖目錄
圖一 分析不同大腸癌期別患者糞便中不同Ribosomal protein基因表現量

37
圖二 利用慢病毒(lentiviral)系統建立穩定表現sh-RPS19細胞株

38
圖三 利用慢病毒(lentiviral)系統降低細胞內RPS19基因表現量

39
圖四 分析細胞中RPS19基因表現下降時處理Oxaliplatin細胞週期量 40
圖五 使用及時定量聚合脢連鎖反應偵測mRNA表現量 41
圖六 使用西方墨點法探討在Oxaliplatin作用下相關蛋白質表現情形 42
圖七 探討RPS19基因表現量下降對於p21蛋白質穩定性的影響 43
圖八 推測RPS19下降對細胞的影響路徑 44
參考文獻

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