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研究生:謝雯琪
研究生(外文):Hsieh wen chi
論文名稱:探討影響細胞生長的基因-CD82和MinichromosomeMaintenance5在前列腺癌中的表現機制
論文名稱(外文):Understanding the Expression of Two Divergent Cell Growth-Regulated Genes, CD82 and Minichromosome Maintenance 5, in the Human Prostate
指導教授:莊宏亨
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:149
中文關鍵詞:前列腺癌生長基因基因轉殖腫瘤標的訊息調控
外文關鍵詞:KAI1MCM5cell proliferationgene regulationprostate
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Minichromosome Maintenance 5 (MCM5) 是屬於MCM家族基因之一員,而MCM蛋白質主要參與染色體複製時之啟發因子 (replication initiation factors)。雖然在前人研究中曾指出前列腺癌患者之尿液沉澱物中含有高量MCM5蛋白質,但MCM5表達於前列腺癌之調控機制仍是未知。CD82又名KAI1,是屬於四級穿膜 (transmembrane 4)家族基因之一員。前人研究中曾將CD82/KAI1視為抑制前列腺癌轉移與參與調控細胞生長中重要的基因。本實驗利用反轉錄聚合連鎖反應與西方點墨法發現MCM5基因可表達於前列腺癌細胞株而且與癌細胞的惡化能力成正相關。相反地, CD82/KAI1的表達可能與癌細胞的轉移能力有相關性。CD82/KAI1之蛋白質表達只有在正常前列腺細胞株 (PZ-HPV-7)與無轉移能力之前列腺癌細胞株 (CA-HPV-10)中被發現,而可轉移之前列腺癌細胞株如LNCaP、PC-3與DU145則無。在低濃度的血清培養液中,大量表現MCM5會促進LNCaP細胞生長,然而,在癌細胞中大量表現CD82/KAI1是會抑制細胞的生長、轉移和入侵的能力。初期動物實驗也證實CD82/KAI1可抑制腫瘤的生長於前列腺癌跟膀胱癌細胞中。也利用siRNA技術證實將CA-HPV-10中的CD82/KAI1剔除會促進細胞的生長、轉移和入侵的能力。實驗成功地建構CD82/KAI1與MCM5基因之報導基因質體 (reporter vector),並利用報導基因表現分析法證實p53及一些抗癌藥物對CD82/KAI1與MCM5基因調控之相異性。相信經由此碩士論文之研究成果,必能進ㄧ步瞭解CD82/KAI1與MCM5之功能基因調控機制,而能於未來應用於臨床醫療檢驗與治療。
The MCM5 is one of the MCM family genes which are replication initiation factors and have been regarded as biological marker of dysplasia and malignancy. The CD82 gene, which is also denoted as KAI1, belongs to a transmembrane 4 superfamily. CD82/KAI1 has been regarded as the metastasis suppressor gene in prostate cancer. Results from RT-PCR and immunoblot assays revealed that gene expression of MCM5 and CD82/KAI1 are relative to prostate neoplasia. Overexpressed MCM5 enhances cell proliferation when cell are cultured in low percentage of serum. However, overexpressed CD82/KAI1 attenuates cell proliferation, migration, and invasion in vitro. Xenograft growth animal study also revealed that CD82/KAI1 exerts antitumorigenic activity on prostate and bladder carcinoma cells in vivo. Knock-down CD82/KAI1 gene expression in the non-metastasis prostate cells significantly enhances in vitro cell proliferation, migration, and invasion. Forced p53 overexpression or anticancer drugs treatments upregulates CD82/KAI1 gene expression; on the contrary, MCM5 gene expression is blocked. Our results demonstrate divergent functions of MCM5 and CD82/KAI1 genes in prostate and bladder cancer cells and determine the regulatory mechanisms of MCM5 and CD82/KAI1 genes.
致謝 v
縮寫對照表 vi
目錄 viii
圖表目錄 xiii
中文摘要 xvi
英文摘要 xvii
壹、緒論 18
一、CD82/KAI1之結構、功能與基因調控之探討 2
二、MCM5之基因調控與功能之探討 8
三、p53之功能與機制 11
四、Nuclear Factor-kappa B (NFκB) 之功能與機制 13
五、Interleukin 6 (IL-6) 對前列腺癌之影響 14
六、雄性素對前列腺之影響 17
七、Adriamycin 19
八、紅酒多酚類物質3,4',5-trihydroxystilbene (Resveratrol) 20
九、17-(Allylamino)-17-demethoxygeldanamycin (17AAG) 21
貳、研究假說與目的 25
ㄧ、探討CD82/KAI1基因於前列腺癌細胞株的功能 26
二、探討MCM5基因於前列腺癌細胞株的功能 26
三、探討轉錄因子與藥物對CD82/KAI1與MCM5基因的調控及訊息的表達 27
參、實驗材料與方法 28
一、細胞培養和化學藥劑 29
二、蛋白質萃取及濃度測量 30
三、西方點墨法 (Immunoblot analysis) 31
四、RNA 萃取 32
五、反轉錄合成反應 (Reverse transcription for synthesis of cDNA) 33
六、聚合酶連鎖反應 (polymerase chain reaction) 34
七、建構CD82/KAI1和MCM5 reporter vector 35
(1) CD82/KAI1 基因報導質體 35
(2)MCM5 基因報導質體 35
八、建構CD82/KAI1表達質體 36
九、建構MCM5表達質體 37
十、建構NFκB報導質體和IκBαm表達質體 37
十ㄧ、建構NIK、雄性素接受體、HSP90基因載體 37
十二、建構CD82/KAI1 siRNA Vector及CD82/KAI1 knock-down 細胞株 38
十三、報導基因表現法 (Transient gene expression Assay) 39
十四、建立穩定性表達特定基因細胞株 40
十五、Cell Proliferation Assay 41
十六、Wound healing assays 43
十七、Matrigel Invasion Assay 43
十八、Xenograft Animal Model 45
十九、統計分析 46
肆、結果 47
一、利用RT-PCR分析CD82/KAI1和MCM5的表現量於前列腺癌細胞 48
二、利用西方點墨法分析CD82/KAI1和MCM5的表現量於前列腺癌細胞 48
三、觀察過度表達MCM5基因對前列腺癌的影響 49
四、p53對MCM5基因的調控 50
五、Adriamycin對MCM5基因的調控 50
六、NFκB對MCM5基因的調控 51
七、Resveratrol對MCM5基因的調控 52
八、17AAG對MCM5基因的調控 52
九、Curcumin對MCM5基因的調控 52
十、雄性素對MCM5基因的調控 53
十ㄧ、觀察過度表達CD82/KAI1基因對前列腺癌的影響 54
十二、Knock-down CD82/KAI1對攝護腺細胞生長影響 55
十三、利用RT-PCR、西方點墨法分析CD82/KAI1的表現量於膀胱癌細胞 56
十四、觀察過度表達CD82/KAI1基因對及膀胱癌細胞的影響 56
十五、p53、Adriamycin對CD82/KAI1基因的調控 57
十六、Resveratrol對CD82/KAI1基因的調控 58
十七、Phorbol ester phorbol-12-myristate-13-acetate (PMA)對CD82/KAI1基因的調控 58
十八、攝護腺細胞株中Cytokines的表現以及影響細胞的生長情形 59
十九、Xenograft growth animal study 60
伍、討論 62
MCM5促進前列腺癌細胞生長 63
Adriamycin及p53會抑制MCM5的表現 64
NFκB調控MCM5的表現 65
抗癌藥物對MCM5的影響 66
R1881抑制MCM5的表現 67
CD82/KAI1抑制前列腺癌細胞生長及轉移 68
Adriamycin及p53會促進CD82/KAI1的表現 70
抗癌藥物對CD82/KAI1的影響 71
IL-6對CD82/KAI1的影響 72
陸、結語 74
柒、附圖 76
捌、附表 103
玖、附錄 106
拾、參考文獻 116
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