Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22871
DC FieldValueLanguage
dc.contributor陳全木zh_TW
dc.contributorChuan-Mu Chenen_US
dc.contributor余長澤zh_TW
dc.contributor.advisor林赫zh_TW
dc.contributor.advisorHo Linen_US
dc.contributor.author黃威寰zh_TW
dc.contributor.authorHuang, Wei-Huanen_US
dc.contributor.other中興大學zh_TW
dc.date2010zh_TW
dc.date.accessioned2014-06-06T07:18:52Z-
dc.date.available2014-06-06T07:18:52Z-
dc.identifierU0005-0807200922584300zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/22871-
dc.description.abstract乳癌 (breast cancer) 是女性發生率最高的癌症,而根據美國癌症協會的統計,乳癌在女性癌症致死率排名僅次於肺癌。文獻指出雌性激素受體 (estrogen receptor alpha, ERα) 活化可促進乳癌的生長,所以在臨床的治療方面ERα陽性的患者可以使用Tamoxifen來抑制ERα活性而獲得較好的預後。Cdk5 (Cyclin-dependent kinase 5) 是一個Ser/Thr 激zh_TW
dc.description.abstractBreast cancer is the most prevalent cancer in women. The statistical information from the American Cancer Society indicates that breast cancer is the second leading cause of death in all cancers. The previous studies show that estrogen receptor alpha (ERα) activation promotes breast cancer cell proliferation. Therefore, ERα antagonist (such as Tamoxifen) was applied as the hormone replacement therapy (HRT) to block ERα activity in patients with ERα protein expression and better prognosis is usually expected. Cdk5 (cyclin-dependent kinase 5), a serine/threonine kinase, belongs to cyclin-dependent kinase family. Cdk5 activity is regulated by its activator protein p35. So far, there is no evidence proves that Cdk5 is involved in cell cycle regulation. Our previous studies demonstrated that Cdk5 activity can induce the phosphorylation of STAT3 at Ser727 and regulate the proliferation of thyroid cancer cells. Furthermore, it has also been demonstrated that the levels of phospho-S727-STAT3 in breast malignant tissues are significantly higher than the normal ones in the patients without ERα protein expression. The purpose of this thesis is to identify the role of Cdk5 in regulating the growth of breast cancer cells. At first, we examined the levels of Cdk5 and p35 proteins in the samples from 28 breast cancer patients provided by Dr. Lee Yueh-Tsung, Chang Bing Show Chwan Memorial Hospital, Taiwan. The results showed that 75 % of patients have higher expression of Cdk5 and 64 % of patients have higher expression of p35 in malignant tissues comparing to normal ones. Cdk5 activity in tumor tissue of one of the patients was testified higher than normal one. The paper demonstrated that Cdk2, which is structurally similar to Cdk5, could phosphorylate ERα and induce its activity. Therefore, the effects of Cdk5 and ERα on proliferation of breast cancer cells become interesting to investigate. Cdk5 and p35 overexpressions were performed in breast cancer cells and cell numbers were evaluated after three-day cultures. The results showed that overexpressions can induce Cdk5 activity and also proliferation of MCF7 cells. In addition, Cdk5 knock-down by shRNA decreased its activity and cell growth after three-day treatments. On the other hand, Cdk5 inhibitor Roscovitine was used to investigate whether ERα activation and subsequent degradation were affected by Cdk5. Besides, the protein interaction between Cdk5 and ERα was studied by co-immunoprecipitation. The results indicated that p35 could stimulate Cdk5 to phosphorylate ERα while the ERα degradation was blocked by proteasome inhibitor MG132. Furthermore, Cdk5-regulated ERα transcriptional activity was also measured by ERE reporter assay. Taken together, Cdk5 might play an important role in regulating ERα function and breast cancer cell proliferation.en_US
dc.description.tableofcontents目錄 中文摘要............................................................i Abstract.............................................................ii 目錄...............................................................iii 圖表目錄............................................................v 第壹章、前言 一、背景.........................................................1 (一)乳房組織介紹...............................................1 (二)乳癌介紹...................................................2 (三)雌性激素及其受體在乳癌中之角色.............................5 (四)Cdk5/p35蛋白之介紹........................................11 (五)實驗模式介紹..............................................13 二、研究動機與目的..............................................14 (一)Cdk5在癌症所扮演的角色....................................14 (二)Cdk和ERα的關係...........................................15 第貳章、材料與方法 一、使用的試劑與廠牌............................................17 二、人類乳癌細胞株的培養........................................19 三、細胞株蛋白質萃取............................................19 四、乳癌病人檢體蛋白質萃取......................................19 五、Dextran-coated charcoal buffer配置..............................20 六、西方墨漬蛋白質表現檢測......................................20 七、免疫沉澱法..................................................21 八、Cdk5激酶活性分析...........................................22 九、細胞生長的測定..............................................22 十、細胞基因轉染技術............................................22 十一、ERE報導基因活性分析........................................23 十二、統計分析....................................................23 第叁章、結果 一、乳癌細胞株的生長曲線及蛋白質表現差異........................24 (一)在完全培養液下觀察乳癌細胞生長曲線........................24 (二)Cdk5和p35在三株乳癌細胞株MCF 7、BT-474和Hs578T 有表現....................................................24 二、乳癌病患檢體中Cdk5與p35蛋白質表現量....................... 25 (一) Cdk5和p35蛋白質在病人檢體中正常組織和腫瘤組織 之比較...................................................25 (二) 病人檢體Cdk5蛋白活性分析................................25 三、Cdk5和p35的過度表現對於乳癌細胞生長的影響..................26 (一)Cdk5和p35的過表現可以促進乳癌細胞MCF 7的生長............26 (二)Rv可以有效抑制乳癌細胞的生長.............................26 (三) ShCdk5和Shp35對於乳癌細胞生長的影響.....................26 四、Cdk5可以藉由磷酸化促進ER轉錄活性而使ERα 蛋白質分解..................................................27 (一)EB可以刺激MCF 7細胞的增生...............................27 (二)EB可以導致ERα蛋白質分解.................................27 (三)Rv可以抑制EB所誘導ERα的蛋白質分解.......................28 (四)Cdk5和ER有蛋白質間交互作用關係...........................28 (五)Cdk5激酶活性可以促進ERα蛋白質水解........................28 (六)MG132抑制ERα的蛋白質分解................................29 (七)過度表現p35活化Cdk5可磷酸化ERα上Ser胺基酸...............29 (八)EB刺激ERα的轉錄活性.....................................30 (九)提升Cdk5激酶活性可刺激ERα的轉錄活性.....................30 第肆章、討論 一、Cdk5活性在乳癌當中扮演重要角色.............................31 二、Cdk5激酶活性對於ERα蛋白之調控............................32 第伍章、結論 圖表目錄 文獻圖示: 附圖一、女性乳房組織剖面.............................................1 附圖二、美國女性癌症死亡人數統計.....................................2 附圖三、民國75年到96年女性癌症死亡人數..............................3 附圖四、雌性激素生成途徑.............................................6 附圖五、ERα的磷酸化受到estrogen結合及活化二級傳訊者的訊息路徑 影響........................................................7 附圖六、ERα和ERβ在女性分布情形.....................................7 附圖七、雌性激素受體的活化機制.......................................8 附圖八、雌性激素類似物會促進老鼠子宮組織的ERα蛋白降解..............9 附圖九、細胞膜上的ERα和細胞核內的ERα訊息傳遞整合模式..............10 附圖十、Cdk5與其他Cdk的差異.......................................12 附圖十一、Cdk5的生理功能...........................................12 附圖十二、p35被切成p25.............................................13 附圖十三、MCF 7細胞株型態..........................................14 附圖十四、BT-474細胞株型態.........................................14 附圖十五、Cdk2/Cyclin A和Cyclin D1活化ER的機制.....................16 附圖十六、Cdk5和其他Cdk家族成員間之親緣關係.......................16 實驗結果: 圖一、三種乳癌細胞株的生長曲線......................................47 圖二、三種乳癌細胞在蛋白質表現上的差異..............................48 圖三、侵入性乳腺管癌invasive ductal carcinoma (IDC) and 原位性腺管癌ductal carcinoma in situ (DCIS) 臨床病人檢體的蛋白質表現................52 圖四、Mucinous adenocarcinoma、Papillary 和 Metaplastic carcinoma 臨床病人 檢體的蛋白質表現.............................................54 圖五、Cdk5和p35蛋白質在乳癌腫瘤組織和正常組織的比較統計..........56 圖六、No. 1病患檢體的.Cdk5蛋白質活性分析...........................57 圖七、過度表現Cdk5或p35可以誘導MCF 7的生長......................58 圖八、Rv可以抑制BT-474乳癌細胞株的生長............................59 圖九、shCdk5和shp35 knockdown 可以抑制BT-474細胞株的生長..........60 圖十、EB 10 nM和EB 100 nM可以刺激MCF 7的生長.....................61 圖十一、Rv可以抑制EB所誘導的ERα蛋白質分解........................62 圖十二、在兩株乳癌細胞株發現Cdk5和ERα有蛋白質間之交互作用.........63 圖十三、在MCF 7細胞中Cdk5激酶活性的增加伴隨ERα蛋白質分解現象....64 圖十四、MG132 可以有效抑制EB所誘導的ERα蛋白質分解現象...........65 圖十五、在MCF 7中過度表現p35刺激Cdk5的活性可以增加p-Ser-ER.......66 圖十六、EB可以刺激ERα的對於ERE的轉錄活性.........................67 圖十七、過度表現p35來增加Cdk5的活性促進ERα對於ERE的轉錄活性....68 圖十八、Cdk5磷酸化ERα來調控ERα的轉錄活性並促進乳癌細胞的生長.....69zh_TW
dc.language.isoen_USzh_TW
dc.publisher生命科學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0807200922584300en_US
dc.subjectCdk5en_US
dc.subject雌性激素zh_TW
dc.subjectERαen_US
dc.subjectbreast canceren_US
dc.subject乳癌zh_TW
dc.titleCdk5可經由活化雌性激素受體alpha促進乳癌細胞株的生長zh_TW
dc.titleCdk5 promotes breast cancer cells proliferation through estrogen receptor alpha activationen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
item.cerifentitytypePublications-
item.grantfulltextnone-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
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