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研究生:莊家驊
研究生(外文):Chia-Hua Chuang
論文名稱:IL-6於攝護腺癌細胞中經由促進p35表現及CDK5活化進而調控血管內皮生長因子的表現
論文名稱(外文):IL-6 increases VEGF expression through regulating p35 expression and CDK5 activity in prostate cancer cells
指導教授:林赫
口試委員:余長澤吳俊錡
口試日期:2017-07-04
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:63
中文關鍵詞:前列腺癌IL-6CDK5p35VEGF
外文關鍵詞:Prostate cancerInterleukin-6CDK5p35VEGF
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週期素激酶5 (Cyclin-dependent kinase 5, CDK5)及其活化蛋白p35在胚胎發育和神經分化過程中佔有重要關鍵。攝護腺癌 (prostate cancer)為雄性激素相關的男性特有內分泌癌症。先前文獻指出CDK5在多種癌症中表現相較正常組織中高,包括在前列腺癌中並促進癌細胞惡化,而CDK5影響腫瘤內皮生長因子 (Vascular endothelial growth factor, VEGF)也被證實藉由穩定HIF-1蛋白所影響。另一方面,細胞因子 (cytokine)對前列腺癌細胞影響很大,其中細胞因子,Interleukin-6 (IL-6)除與免疫發炎相關外,在神經系統中刺激p35表現及CDK5活化造成神經相關病變,且IL-6在癌症中被認為與促進惡化及血管新生相關。但在前列腺癌中IL-6刺激是否會因CDK5活化來影響VEGF進行調控仍待進一步的探討。本研究結果顯示,在前列腺癌細胞中IL-6刺激VEGF表現可透過Erk/Egr1影響p35表現及CDK5活化產生。實驗利用抑制劑抑制Erk活性後,Egr1、p35及VEGF蛋白表現減少。而在IL-6處理下發現細胞核中Egr1、p35蛋白分布表現增加及CDK5活性增加情形,並在抑制CDK5活性後VEGF表現跟著減少。綜合以上結果顯示IL-6在前列腺癌中促進VEGF表現為透過CDK5活化的參與,此發現顯示IL-6刺激對於誘導前列腺癌血管新生進一步的可能性,並說明CDK5在此間扮演著重要角色。期望此研究結果對於癌症預測及治療提供新的方向。
目錄
第壹章、前言 1
一、背景 1
(一)、攝護腺與攝護腺癌的介紹 1
(二)、Interleukin-6的介紹 3
(三)、CDK5的介紹 6
(四)、VEGF與Angiogenesis的介紹 9
(五)、IL-6與CDK5間的關係 14
(六)、IL-6與VEGF及CDK5與VEGF間關係 14
二、研究動機與目的 16
第貳章、材料與方法 18
一、細胞培養 (Cell Culture) 18
二、蛋白質定量與定性分析 19
(一)、蛋白質萃取 (Protein extraction) 19
(二)、蛋白質濃度測定 (Bradford assay) 19
三、西方墨點法 (Western Blot) 20
(一)、聚丙烯醯胺膠體電泳 (SDS-polyacrylamide gel electrophoresis) 20
(二)、免疫墨點法 (Immunoblotting, IB) 20
四、細胞蛋白核質分離 (Cellular protein fractionation) 22
五、質體轉染技術 (Plasmid transfection) 23
六、慢病毒轉導 (Lentiviral transduction) 24
(一)、慢病毒生產 (Lentiviral production) 24
(二)、慢病毒感染 (Lentiviral infection) 24
七、離體激酶活性測定 (In Vitro kinase assay) 25
八、分泌至細胞外的蛋白測定 (Secretion experiment) 25
九、藥物使用 (Drugs) 26
十、量化分析 (Quantify) 26
十一、統計分析 (Statistics) 26
第參章、實驗結果 27
一、攝護腺癌中IL-6刺激下觀察對於VEGF蛋白質表現影響 27
二、觀察IL-6誘導VEGF表現增加的可訊息傳遞路徑 27
三、觀察Erk活性對於VEGF蛋白表現的影響 28
四、IL-6刺激下Egr1參與影響VEGF蛋白表現 28
(一)、探討IL-6處理下細胞核/質中Egr1、p35分布表現情形 29
(二)、降低Egr1蛋白表現量影響細胞內p35、VEGF蛋白表現 29
(三)、降低Egr1蛋白表現量導致減少IL-6所誘導的VEGF蛋白表現 29
五、IL-6刺激下CDK5活性影響VEGF蛋白表現 30
(一)、IL-6刺激處理下抑制劑抑制CDK5活性觀察對VEGF蛋白表現的影響 30
(二)、降低CDK5蛋白表現量影響細胞內VEGF蛋白表現 30
(三)、降低CDK5蛋白表現量導致減少IL-6所誘導的VEGF蛋白表現 31
(四)、IL-6刺激處理下CDK5突變體抑制CDK5活性觀察對VEGF蛋白表現的影響 31
(五)、In vitro kinase assay證實IL-6刺激增加CDK5活性 32
六、IL-6刺激下誘導攝護腺癌細胞分泌VEGF蛋白至細胞外 33
第肆章、結果與討論 34
一、IL-6對於VEGF的調控影響 34
二、IL-6與CDK5在攝護腺癌中的關係 34
三、IL-6可能影響PARP的蛋白表現增加 35
四、IL-6透過CDK5誘導VEGF表現 35
五、IL-6透過Erk/Egr1/p35使CDK5活化進而誘導VEGF分泌表現 36
第伍章、結論 37
第陸章、參考文獻 38
第柒章、實驗結果圖 48
圖表目次
附圖一、攝護腺癌轉移位置圖 2
附圖二、1979-2013台灣攝護腺癌年齡發生率統計資料圖 2
附圖三、IL-6訊號組成系統示意圖 4
附圖四、IL-6對於生理反應的多種功能 5
附圖五、IL-6與癌症的關係 5
附圖六、CDK5在神經系統生理調控 6
附圖七、CDK5參與癌症中的機制 8
附圖八、不同型之VEGF與VEGFR結合下游作用 11
附圖九、腫瘤血管新生機制示意圖 13

附表一、VEGF家族蛋白各成員功能 10
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