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研究生:張耀云
研究生(外文):Yao-Yun Jhang
論文名稱:胺基胜肽酶A在大腸癌轉移所扮演的角色
論文名稱(外文):The role of aminopeptidase A in the metastasis of colon cancer
指導教授:黃智生黃智生引用關係
指導教授(外文):Chih-Sheng Huang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:56
中文關鍵詞:胺基胜&胺基胜&胺基胜&胺基胜&胺基胜&
外文關鍵詞:aminopeptidase A
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結腸直腸癌(CRC)在台灣以及全世界是第三常見的癌症,大約有將近有半數患結腸直腸癌的患者可以藉由手術與抗癌藥物的合併治療達到治癒的目的,但是癌症轉的轉移往往會造成治療失敗。我們使用微陣列分析的方法分析不同時期結腸直腸癌患者的基因表現,藉由比對原發性腫瘤比正常組織之間有差異表現的基因,再把這些基因與轉移性腫瘤之間基因表現有差異的基因,做交集我們一共得到157個可能與癌症轉移相關的基因,再利用RT-PCR與real-time PCR去驗證157個當中還沒有文獻報導與結腸直腸癌轉移有直接關係的11個基因,而截至目前為止有關於ENPEP這個基因與結腸直腸癌的關係還並不清楚,故我們最後集中在胺基胜肽酶A﹙ENPEP﹚上的研究。

當使用real-time PCR分析不同時期臨床病人的檢體,我們發現到ENPEP基因的表現會隨者臨床時期的增加而變多,這個結果顯示ENPEP的表現可能會因為癌症的生成而有所增加,而在轉移時期ENPEP的表現雖有下降,但也是與原發性腫瘤的ENPEP表現量有所差異。為了研究ENPEP在結腸直腸癌中癌轉移的角色,我們將ENPEP這個基因大量表現於內生性蛋白很少的結腸直腸癌細胞株(SW480)中,結果發現ENPEP最佳的表現時間為轉染後24至48小時之間。接著,我們利用wound healing和細胞侵襲分析去證明當ENPEP在結腸直腸癌細胞中大量表現時是否會增強癌細胞的移動力和侵襲能力,實驗結果發現與vector control相比,細胞表現ENPEP的確會有較多穿過transwell底部及較快把間隙填滿,另外我們也發現到在大量表現ENPEP的SW480有較高twist的表現,real-time PCR的結果發現在大量表現ENPEP的SW480較具有幹細胞的特質,也發現NF-κb及活化AKT表現量提升。綜合上述實驗的結果,我們推測大量表現ENPEP的SW480會增進其移動及侵襲能力也許是藉由twist所主導的訊號傳遞,且ENPEP的表現應該有助於結腸直腸癌的癌轉移。

Colon cancer is the third most common cancer worldwide. About half of patients with colorectal cancer can be cured by surgery and multimodal treatment. However, metastasis is the most important cause for cure failure. We used microarray to analyze the gene expression profiles of colorectal cancer specimens. Among the 157 metastasis-related genes, RT-PCR and real-time PCR were used to verify 11 of them. However, we focused on glutamyl aminopeptidase (Aminopeptidase A) (ENPEP) because its relationship with colon cancer is unknown.
Based on real-time PCR results from different stages of patients, we found that the expression levels of ENPEP are higher in those with more advanced tumor grade. We also overexpressed ENPEP in SW480 and found that the number of invading cells crossing coated matrigel was more than that of vector control. ENPEP may induced EMT transcription factors like Snail,Slug or Twist.By using Q-PCR to detect the amount of transcription factors, we found that Twist expression is about four folds more than that in the vector control. To study the stemness gene profile, we check the expressions of Nanog, Sox2, Oct4 and C-myc and found significant increase in the expression of these gene. Combined, our data suggest that ENPEP may be involved in enhancement of mobility, invasion and metastatic capabilities of colon cancer cells through the Twist mediated signaling pathways.

論文電子檔著作權授權書 i
論文電子檔審定證明書 ii
論文審定同意書 iii
致謝 iv
中文摘要 vi
英文摘要 vii
目錄 viii
第一章 緒論 1
第一節 結腸直腸癌(Colorectal cancer;CRC) 1
第二節 胺基胜肽酶A(Aminopeptidase A;ENPEP)簡介 4
第三節 癌症的轉移(Metastasis) 5
第四節 研究動機與目的 6
第二章 材料與方法 7
第一節 實驗材料 7
1. 勝任細胞株(Competent cell strain) 7
2. 細胞株(Cell lines) 7
3. 質體(Plasmids) 8
4. 抗體(Antibodies) 8
5. 實驗套組(Kits) 8
6. 細胞培養液(Culture medium) 9
7. DNA電泳之用 9
8.西方墨點轉漬檢視之用………………………………………………...10
9.引子清單………………………………………………………………...10
第二節 實驗方法………………………………………………………………11
1. 總核醣核酸萃取(Total RNA extraction from cell line)……………11
2. 反轉錄聚合酶鏈鎖反應(Reverse transcription polymerase chain reaction;RT-PCR)…………………………………………………12
3. 聚合酶鏈鎖反應(Polymerase chain reaction;PCR)……………12
4. 即時聚合酶鏈鎖反應(Real-time polymerase chain reaction;RT-PCR) 13
5. ENPEP基因表現質體的建構 13
5.1 ENPEP基因片段放大………………………………………………..13
5.2 PCR產物之凝膠萃取純化(Gel extraction of PCR product)……14
5.3 PCR產物與表現載體之接合﹙DNA ligation﹚……………………..14
5.4轉形作用(Transformation)………………………………………..15
5.5菌落聚合酵素連鎖反應(Colony PCR)……………………………15
6. 小量質體DNA之萃取(Mini plasmid DNA extraction) 16
7. 中量質體DNA之萃取(Midi plasmid DNA extraction) 16
8. 酒精沉澱(Ethanol precipitation) 18
9. 短暫性轉染作用(Transient transfection) 18
10. 慢病毒之製備(Lentivirus production) 18
11. 慢病毒之感染(Lentivirus infection) 19
12. 西方墨點法(Western blot) 19
13. 細胞移動分析(Migration assay)…………………………………22
14. 細胞侵襲分析(Invasion assay) 22
15. Stable cell lines 23
16. NF-κb reporter assay 23
第三章 實驗結果 25
1. 藉由Real-time PCR直接分析不同時期臨床病人的檢體 25
2. 利用Real-time PCR與Western blot找出最適合表現ENPEP之細胞株 25
3. 構築ENPEP大量表現之質體及利用Western blot找出最適合表現ENPEP時間點 26
4. 利用Transwell進行細胞移動力的分析 26
5. 利用wound healing進行細胞移動力的分析 26
6. 利用細胞侵襲分析研究細胞的侵襲能力 27
7. 利用西方點墨法分析研究EMT marker的變化…………………………27
8. 利用Real-time PCR與Western blot分析調控EMT的transcription factors………………………………………………………………………28
9. 利用Real-time PCR分析Stem cell marker的變化……………………..28
10. 利用NF-κb reporter assay分析NF-κb活性變化……………………….29
11. 利用西方點墨法分析AKT-P的表現………………………………………29
12. 利用Real-time PCR與Western blot找出最適合konckdown ENPEP表現之clone…………………………………………………………………..30
13. 利用細胞侵襲分析研究細胞的侵襲能力………………………………….30
14. 構築ENPEP永久表現之質體及利用RT-PCR和Western blot分析ENPEP之表現…………………………………………………………….31
15. 利用細胞侵襲分析研究細胞的侵襲能力………………………………….31
第四章 實驗討論……………………………………………………………………32
第五章 參考文獻……………………………………………………………………37
第六章 實驗結果圖表………………………………………………………………42


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