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研究生:陳宣余
研究生(外文):Hsuan-Yu Chen
論文名稱:探討與PTPN2有交互作用的蛋白,以及在肺癌上所扮演的角色
論文名稱(外文):Study on PTPN2 interacting protein and its role in lung cancer
指導教授:陳健尉陳健尉引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:94
中文關鍵詞:酪胺酸去磷酸酶非接受器第二型酵母菌雙雜合技術
外文關鍵詞:protein tyrosine phosphatase non-receptor type 2yeast two hybrid system
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根據行政院衛生署九十五年的統計資料顯示,肺癌為台灣地區惡性腫瘤首要死亡原因,其中又以肺腺癌最為常見。通常癌症是因為細胞的基因變異所引起的,這些基因包含致癌基因及抑癌基因。在細胞中,磷酸化和去磷酸化蛋白扮演了重要角色,其可藉由活化不同的蛋白來調控細胞內各項反應,包含細胞內的生長、代謝、分化作用等,全面性的影響細胞中各項生理機制。本研究的目的,是利用酵母菌雙雜合系統(yeast two hybrid system)找出與酪胺酸去磷酸酶非接受器第二型(protein tyrosine phosphatase non-receptor type 2,PTPN2)有交互作用的蛋白質。 PTPN2屬於PTP(protein tyrosine phosphatase)superfamily中的一員,而PTP是一種細胞訊息分子,利用訊息傳遞調控酪胺酸去磷酸化。PTP可調控細胞內多項反應,包含:生長、增殖、分化、遷徙、代謝、存活。在本研究中,已經利用酵母菌雙雜合技術,從人類肝臟cDNA基因庫中,篩選出八個與PTPN2有交互作用的蛋白質。前人研究發現,CD74(major histocompatibility complex class II invariant chain)及PRDX6(Peroxiredoxin 6)會促使細胞增生。因此,挑選CD74及PRDX6做後續實驗。利用免疫共沉澱法(Co-immunoprecipi tation),也證實兩者之間有交互作用。此外,也藉由大量表現PTPN2蛋白以觀察對細胞行為的影響。經由免疫螢光染色實驗,發現PTPN2分佈在細胞的內質網。根據MTT試驗、細胞貼附性生長(anchorage-dependent growth)及細胞非貼附性生長(anchorage-independent growth)實驗結果發現,PTPN2會促進肺癌細胞CL1-0及H1299的細胞增生,並且造成細胞內磷酸化的ERK增加。然而,PTPN2對CL1-0、CL1-5及H1299細胞的侵襲能力及遷移能力並沒有顯著的影響。因此我們推測,PTPN2會藉由調控磷酸化的ERK,而造成肺癌細胞增生的現象。這個推測仍需做進一步的研究與探討,詳細的作用機制也需後續實驗加以證明。藉由此研究我們可以提供一個調控肺癌細胞增生的機制,或許在未來可以應用在癌症治療上。
According to statistical data in 2006 of Department of Health, the lung cancer is the main reason of death about malignant tumor in Taiwan, especially, adenocarcinomas is the most common one. Cancer is often caused by gene mutation, including oncogenes and tumor suppressor genes. Protein modification, such as phosphorylation and dephosphorylation, plays an important role to regulate many cellular responses including growth, metabolism and differentiation by activating different proteins. The aim of this study is to find which proteins interacting with PTPN2 (protein tyrosine phosphatase non-receptor type 2) by yeast two hybrid assay. PTPN2, belongs to PTP (protein tyrosine phosphatase) superfamily, is a signal transduction protein and regulates protein tyrosine phosphorylation through signal transduction pathway. PTP controls a diverse array of cellular responses including growth, proliferation, differentiation, migration, metabolism and survival. In this study, we utilized yeast two hybrid assay, and identified eight PTPN2-interacting proteins from human liver cDNA library. Previous work showed that CD74 (major histocompatibility complex class II invariant chain) and PRDX6 (Peroxiredoxin 6) could promote cell proliferation. Therefore, CD74 and PRDX6 were demonstrated in this study, and they are indeed confirmed to interact with PTPN2 by using co-immunoprecipitation. Additionally, the cell functions were also assayed under PTPN2 over- expression in cell. By immunofluorescent staining, we found that PTPN2 located in endoplasmic reticulum (ER). Overexpression of PTPN2 could significantly promote lung cancer cell proliferation by MTT assay, anchorage-dependent growth and anchorage-independent growth and increase the expression of phospho-ERK. However, PTPN2 had no effect on invasive and migrative ability. Taken together, we speculated that PTPN2 might promote lung cancer cell proliferation through ERK phosphorylation, and it needed more efforts to study in the future. By means of this study we provided a putative mechanism of modulating lung cancer cell proliferation, which might benefit to the development of target therapy in the future.
中文摘要 .................................................i
英文摘要 ................................................ii
目次 ...................................................iii
表及附表目次 ............................................vi
圖及附圖目次 ...........................................vii
縮寫字對照表 ............................................ix
第一章 序論
第一節、文獻回顧 .........................................1
一、肺癌 .................................................1
二、癌轉移 ...............................................3
三、蛋白質酪胺酸去磷酸化 .................................4
四、蛋白質酪胺酸去磷酸酶 .................................5
五、蛋白質酪胺酸去磷酸酶與癌症 ...........................6
六、蛋白質酪胺酸去磷酸酶非接受器第二型 ...................7
第二節、研究動機與架構 ...................................7
第二章 材料與方法
第一節、材料 ............................................10
第二節、細胞株及細胞培養 ................................10
第三節、細胞冷凍保存及解凍 ..............................11
第四節、細胞蛋白質之萃取 ................................11
第五節、蛋白質定量 ......................................12
第六節、免疫共沉澱 ......................................12
第七節、蛋白質凝膠電泳分析 ..............................13
第八節、孔雀藍染色 ......................................13
第九節、西方墨點法 ......................................14
第十節、細胞RNA萃取 .....................................15
第十一節、反轉錄聚合酶連鎖反應 ..........................16
第十二節、DNA片段回收之純化 .............................16
第十三節、細胞質體DNA萃取 ...............................17
第十四節、DNA黏接反應 ...................................18
第十五節、勝任細胞製備 ..................................18
第十六節、轉形作用 ......................................18
第十七節、菌種甘油保存法 ................................19
第十八節、菌落聚合酶連鎖反應 ............................19
第十九節、大量質體萃取 ..................................19
第二十節、轉染作用 ......................................20
第二十一節、穩定表現細胞株加藥篩選 ......................21
第二十二節、噻唑藍試驗 ..................................21
第二十三節、細胞侵襲能力分析 ............................22
第二十四節、細胞遷移能力分析 ............................23
第二十五節、軟瓊脂中細胞聚落形成分析 ....................23
第二十六節、細胞聚落形成分析 ............................24
第二十七節、免疫螢光染色 ................................24
第二十八節、酵母菌雙雜合系統 ............................25
第二十九節、統計分析方式 ................................30
第三章 實驗結果
第一節、PTPN2在肺癌細胞中的表現效率 .....................31
第二節、PTPN2基因構築 ...................................31
第三節、PTPN2進行穩定表現細胞株加藥篩選 .................31
第四節、大量表現PTPN2對肺癌細胞增生能力的影響 ...........31
第五節、大量表現PTPN2對肺癌細胞貼附性生長的影響 .........32
第六節、大量表現PTPN2對肺癌細胞非貼附性生長的影響 .......32
第七節、大量表現PTPN2對肺癌細胞侵襲能力的影響 ...........33
第八節、大量表現PTPN2對肺癌細胞遷移能力的影響 ...........33
第九節、觀察PTPN2在細胞內的分佈情形 .....................33
第十節、大量表現PTPN2對肺癌細胞磷酸化的影響 .............34
第十一節、分析與PTPN2有交互作用的蛋白 ...................35
第十二節、酵母菌雙雜合系統 ..............................35
第四章 討論 .............................................36
第五章 結論 .............................................43
參考文獻 ................................................44
實驗結果表圖 ............................................50
附表與附圖 ..............................................72
附錄 ....................................................85
行政院衛生署九十五年死因統計
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