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dc.contributorJeremy J.W. Chenen_US
dc.contributor.authorYen-Chun Chenen_US
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dc.description.abstractProtein phosphorylation is crucial for carcinoma progression. PP1-β plays a dual rule as a tumor suppressor gene and an oncogene in different cancer types. PP1-β inhibits the tumorigenic ability of malignant melanoma and breast cancer, but it promotes cancer progression in chronic lymphocytic leukemia. The role of PP1-β in non-small cell lung carcinoma is still unknown. Therefore, we investigate the rule of PP1-β and the relation between PP1-β and HLJ1-NPM1 interaction. In our study, the expression of PP1-β in CL1-0 and A549 is higher than in CL1-5 cell line. Silencing PP1-β in CL1-0 and A549 cells inhibit cell invasion, migration, proliferation and colony formation. We discovered that silencing PP1-β down-regulates the binding ability of AP-2α to MMP-2 promoter and also enhance MMP-2 activity. However, silence of PP1-β enhances the interaction of HLJ1 and NPM1, but it is not the consequence of phosphoNPM1 at T199 by PP1-β. Moreover, silence of PP1-β does not change NPM1 distribution which modified the cell cytoskeleton. In conclusion, PP1-β plays an important role in non-small cell lung carcinoma.en_US
dc.description.abstract蛋白質的磷酸化對蛋白質在癌症的進程中是很重要的調控機轉, PP1家族調控大部分蛋白質的去磷酸化,其中PP1-β同時具有致癌基因以及抑癌基因兩種相反的角色:在黑色素瘤以及乳癌中是扮演著抑癌基因的角色,而在慢性淋巴白血病中卻是扮演著致癌基因的角色。但是PP1-β在肺癌中的角色卻還沒被定義,因此本研究是探討非小細胞肺癌中PP1-β的角色以及PP1-β對HLJ1-NPM1的相關機轉。在本研究中,PP1-β在CL1-0以及A549的表現量比CL1-5高,因此我們將PP1-β在CL1-0 以及A549肺癌細胞株中靜默,發現靜默PP1-β會抑制非小細胞肺癌的侵襲、移動、增生以及聚落形成的能力。在PP1-β相關機轉方面,我們使用染色質免疫沉澱以及膠質?譜實驗發現到靜默PP1-β會使得AP-2α對於MMP-2的啟動子結合能力下降而MMP-2的活性上升。我們又透過免疫共沉澱實驗及西方墨點法實驗觀察到靜默PP1-β能使NPM1和HLJ1的交互作用能力上升,但是卻和PP1-β對NPM1的T199位點磷酸化沒有直接的影響。也透過免疫螢光染色實驗觀察到PP1-β的靜默對NPM1的分布沒有影響,但是會使細胞骨架改變。由以上結果可以得知PP1-β對於非小細胞肺癌進程扮演著重要的角色。zh_TW
dc.description.tableofcontents中文摘要------------------------------------------------------------- i Abstract--------------------------------------------------------------- ii 目錄------------------------------------------------------------------- iii 實驗結果圖表目錄------------------------------------------------- iv 縮寫字對照表------------------------------------------------------- viii 第一章、緒論------------------------------------------------------ 1 第一節 前人研究----------------------------------------------------- 1 一、肺癌------------------------------------------------------------- 1 (一)肺癌的成因及症狀----------------------------------------- 1 (二)肺癌的分類------------------------------------------------- 2 (三)癌症轉移--------------------------------------------------- 3 二、PP1-β 蛋白----------------------------------------------------- 3 (一)PP1家族---------------------------------------------------- 3 (二)PP1-β 的功能及相關機轉--------------------------------- 4 (三)PP1-β 與癌症的關係--------------------------------------- 5 三、NPM1 蛋白----------------------------------------------------- 6 (一)NPM1的功能----------------------------------------------- 6 (二)NPM1與癌症的關係--------------------------------------- 7 第二節 研究目的及研究架構圖-------------------------------------- 8 第二章、實驗材料與方法------------------------------------------- 10 第一節 材料----------------------------------------------------------- 10 第二節 細胞株及細胞培養------------------------------------------- 10 一、細胞株----------------------------------------------------------- 10 二、細胞培養-------------------------------------------------------- 10 第三節 細胞冷凍保存及細胞解凍----------------------------------- 11 一、細胞冷凍保存--------------------------------------------------- 11 二、細胞解凍-------------------------------------------------------- 11 第四節 轉染作用----------------------------------------------------- 12 第五節 慢病毒屬感染------------------------------------------------ 12 第六節 加藥篩選建立穩定表現細胞株------------------------------ 13 第七節 聚合?連鎖反應---------------------------------------------- 13 第八節 DNA片段回收之純化與黏接反應--------------------------- 14 一、片段回收純化--------------------------------------------------- 14 二、黏接反應-------------------------------------------------------- 14 第九節 製備勝任細胞與轉型作用----------------------------------- 15 一、製備勝任細胞--------------------------------------------------- 15 二、轉型作用-------------------------------------------------------- 15 第十節 菌種甘油保存法---------------------------------------------- 16 第十一節 菌落聚合?連鎖反應-------------------------------------- 16 第十二節 大量質體萃取---------------------------------------------- 16 第十三節 細胞蛋白質之萃取與定量--------------------------------- 17 一、細胞蛋白質之萃取---------------------------------------------- 17 二、蛋白質定量------------------------------------------------------ 18 第十四節 免疫沉澱法------------------------------------------------ 18 第十五節 蛋白質凝膠電泳分析-------------------------------------- 19 第十六節 西方墨點法------------------------------------------------ 19 第十七節 細胞侵襲能力分析----------------------------------------- 20 第十八節 細胞遷移能力分析----------------------------------------- 21 一、傷口癒合試驗--------------------------------------------------- 21 二、透孔試驗-------------------------------------------------------- 21 第十九節 細胞聚落形成分析----------------------------------------- 22 第二十節 細胞存活試驗--------------------------------------------- 22 第二十一節 免疫螢光染色------------------------------------------- 23 第二十二節 染色質免疫沉澱分析----------------------------------- 23 第二十三節 即時定量PCR分析-------------------------------------- 24 第二十四節 條件培養液的製備-------------------------------------- 24 第二十五節 膠質?譜分析------------------------------------------- 25 第二十六節 統計分析------------------------------------------------ 25 第三章、實驗結果--------------------------------------------------- 26 第一節 在CL1-0及A549細胞株中PP1-β 的表現量比較多-------- 26 第二節 在CL1-0肺癌細胞株中建立了PP1-β基因靜默的穩定細胞株-------------------------------------------------------- 26 第三節 在A549肺癌細胞株中建立了PP1-β基因靜默的穩定細胞株--------------------------------------------------------- 27 第四節 CL1-0穩定靜默PP1-β細胞株的細胞侵襲能力下降------ 27 第五節 A549穩定靜默PP1-β細胞株的細胞侵襲能力下降------- 27 第六節CL1-0穩定靜默PP1-β細胞株的細胞移動能力下降------ 28 第七節 A549穩定靜默PP1-β細胞株的細胞移動能力下降------- 28 第八節 CL1-0穩定靜默PP1-β細胞株的細胞群落形成能力下降 29 第九節 A549穩定靜默PP1-β細胞株的細胞群落形成能力下降- 29 第十節 CL1-0穩定靜默PP1-β細胞株的細胞增生能力差異不大 29 第十一節 A549穩定靜默PP1-β細胞株的細胞增生能力下降---- 30 第十二節 CL1-0穩定靜默PP1-β細胞株中NPM1在T199位點磷酸化的表現量降低-------------------------------------- 30 第十三節 CL1-0穩定靜默PP1-β細胞株中HLJ1-NPM1交互作用能力上升------------------------------------------------- 30 第十四節 CL1-0穩定靜默PP1-β前後NPM1的皆分布於核仁--- 31 第十五節 CL1-0穩定靜默PP1-β細胞株中AP-2α對MMP-2啟動子結合能力稍微下降------------------------------------ 31 第十六節 CL1-0穩定靜默PP1-β細胞株中MMP-2的活性上升--- 31 第十七節 CL1-0穩定靜默PP1-β的細胞株中AKT蛋白表現量降低且A549穩定靜默PP1-β的細胞株中cyclin D1蛋白表現量上升--------- 32 第四章、 討論------------------------------------------------------ 33 第五章、 結論------------------------------------------------------ 38 實驗結果圖表目錄 圖一、在不同的肺癌細胞株中觀察PP1-β的表現量------------------ 39 圖二、在CL1-0將PP1-β穩定基因靜默------------------------------- 40 圖三、在A549將PP1-β穩定基因靜默-------------------------------- 41 圖四、CL1-0 穩定靜默PP1-β細胞株中細胞侵襲能力--------------- 42 圖五、A549 穩定靜默PP1-β細胞株中細胞侵襲能力---------------- 43 圖六、CL1-0 穩定靜默PP1-β細胞株中細胞移動能力--------------- 44 圖七、A549 穩定靜默PP1-β細胞株中細胞移動能力---------------- 45 圖八、CL1-0穩定靜默PP1-β細胞株中細胞移動能力---------------- 46 圖九、A549穩定靜默PP1-β細胞株中細胞移動能力----------------- 47 圖十、CL1-0 穩定靜默PP1-β細胞株中細胞聚落形成能力---------- 48 圖十一、A549穩定靜默PP1-β細胞株中細胞聚落形成能力--------- 49 圖十二、CL1-0穩定靜默PP1-β細胞株中細胞增生能力------------- 50 圖十三、A549穩定靜默PP1-β細胞株中細胞增生能力-------------- 51 圖十四、CL1-0穩定靜默PP1-β細胞株的NPM1蛋白質在T199胺基酸的磷酸化能力------------------------------------------ 52 圖十五、CL1-0穩定靜默PP1-β細胞株的HLJ1-NPM1交互作用能力------------------------------------------------------------ 53 圖十六、CL1-0穩定靜默PP1-β細胞株的NPM1分布---------------- 54 圖十七、CL1-0穩定靜默PP1-β細胞株的NPM1分布---------------- 55 圖十八、CL1-0穩定靜默PP1-β細胞株的NPM1分布---------------- 56 圖十九、CL1-0穩定靜默PP1-β細胞株的AP-2α對MMP-2啟動子的結合能力------------------------------------------------- 57 圖二十、CL1-0穩定靜默PP1-β細胞株的MMP-2活性--------------- 58 圖二十一、CL1-0及A549穩定靜默PP1-β細胞株的PP1-β相關蛋白分析--------------------------------------------------- 59 圖二十二、pEGFP-C3載體-------------------------------------------- 60 表一、相關引子序列表格--------------------------------------------- 61 附錄一---------------------------------------------------------------- 62 參考文獻-------------------------------------------------------------- 65zh_TW
dc.subjectlung canceren_US
dc.titleThe study of functional role of protein phosphatase 1β in tumor progression of non-small cell lung carcinomaen_US
dc.typeThesis and Dissertationen_US
item.fulltextwith fulltext-
item.openairetypeThesis and Dissertation-
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