14-3-3蛋白是一種molecular chaperones。它們幫助調控有關細胞繁殖、分化，以及存活的訊息傳遞途徑。它們直接或間接與繁殖訊號傳遞蛋白，如PKC，MEK kinases，PI3-K和Raf有關連。在人類，有七個不同的14-3-3基因：β（beta）、γ（gamma）、ε（epsilon）、η（eta）、σ（sigma）、τ/θ（tau/theta）、ζ（zeta）以及一些可能的假基因，而酵母菌有2個和植物有15個14-3-3基因。雖然有基因歧異性，但是所有14-3-3 isotypes有很多相同且保守之序列。
在現有的研究顯示，在所有14-3-3的家族當中，14-3-3 sigma被認為和癌症的發生有最直接的關係，因為目前它的功能被認為是抑制細胞週期之細胞增生的腫瘤抑制者。而在許多腫瘤的發生中，14-3-3 sigma有很高的比率是不活化的；更重要者，在大多乳癌細胞中14-3-3 sigma不表現。因為14-3-3 sigma藉由在細胞質中，結合上cyclin E-CDK2複合物與cyclin B-CDC2複合物使之不活化並留在細胞質中，所以使細胞週期分別停留在G1與G2。而其餘14-3-3 isotypes在乳癌形成的角色亦無定論。
本實驗的目的是要在人類非腫瘤的乳房組織與乳房腫瘤組織之中，尋找14-3-3 gamma表現量的差異。經由反轉錄聚合酶連鎖反應、蛋白質西方墨漬法分析、免疫組織染色與即時定量聚合酶連鎖反應等實驗初步發現14-3-3 gamma在乳房腫瘤組織中DNA、RNA及蛋白質的層面上有過量表現，至於14-3-3 gamma可能在乳房腫瘤發生中所扮演的角色，則有待後續之研究。

The chaperone proteins designated 14-3-3 are expressed in all eukaryotic cells; they help to regulate signal transduction pathways controlling proliferation, differentiation, and survival. They associated directly or indirectly with proliferative signal-transducing proteins such as PKC, MEK kinases, PI3-kinase and Raf. In human, there are seven isotypes of 14-3-3 genes: β（beta）、γ（gamma）、ε（epsilon）、η（eta）、σ（sigma）、τ/θ（tau/theta） andζ（zeta）, some of which would be pseudogenes, and yeast and plant each have two and fifteen genes. Althought these genes are diverse, all 14-3-3 isotypes share many conservation domains in amino acid sequences.
The previous studies have suggested that 14-3-3 sigma is most directly linked to cancer because it is thought to function as a tumor suppressor by inhibiting cell-cycle progression. In tumor formation, inactivation of 14-3-3 sigma occurs with high frequency. More importantly, expression of 14-3-3 sigma is silenced in most breast cancer cells. The 14-3-3 sigma protein is associated with cyclin E-CDK2 complex as well as cyclin B-CDC2 complex and mediated their inactivation by cytoplasmic localization and causing cell-cycle arrest in G2 and G1. However, the roles of other 14-3-3 isotypes in the formation of breast cancer are controversial in published reference.
The aim of this study was to determine the differential expressions of 14-3-3 gamma in non-tumor tissues and corresponding tumor tissues. Amplification and overexpression of 14-3-3 gamma in DNA, RNA, and protein of breast tumor tissues were found by experiments of RT-PCR, Western blot analysis, immunohistochemistry and Real-time PCR. However, the role of 14-3-3 gamma in the formation of breast cancer requires further study.

中文摘要------------------------------------------1
英文摘要------------------------------------------3
緒 論------------------------------------------5
乳癌--------------------------------------------5
近年來乳癌分子生物學的研究----------------------------7
14-3-3 Protein--------------------------------------10
14-3-3 Protein theta/tau subtype---------------------------14
14-3-3 Protein eta subtype------------------------------14
14-3-3 Protein epsilon subtype----------------------------15
14-3-3 Protein beta subtype------------------------------15
14-3-3 Protein zeta subtype------------------------------16
14-3-3 Protein sigma subtype-----------------------------16
14-3-3 Protein gamma subtype----------------------------17
實驗目的-----------------------------------------19
材料與方法---------------------------------------21
檢體與玻片--------------------------------------21
Total RNA萃取------------------------------------21
RT-PCR----------------------------------------22
定序與序列比對-----------------------------------23
Western blot analysis---------------------------------23
免疫組織化學染色----------------------------------25
Genomic DNA的純化--------------------------------26
Real-time quantitative PCR-----------------------------27
實驗結果-----------------------------------------29
RT-PCR檢測臨床乳癌成對檢體間基因表現------------------29
PCR產物定序結果與生物資訊資料庫基因比對----------------29
臨床乳癌成對檢體間蛋白質表現-------------------------29
免疫組織化學染色分析-------------------------------30
乳癌成對檢體基因組DNA 14-3-3 gamma基因之copy number-------30
討 論-----------------------------------------31
參考資料-----------------------------------------36
表格與附圖---------------------------------------43




圖表目錄

圖一：人類14-3-3七種 isotypes的胺基酸序列----------------43
圖二：14-3-3蛋白調節基因表現--------------------------44
圖三：14-3-3蛋白調節細胞週期--------------------------45
圖四：14-3-3蛋白homodimers結構------------------------46
圖五：14-3-3蛋白參與細胞Apoptosis的過程-----------------47
圖六：Real-time quantitative PCR所設計的primer-------------48
圖七：臨床乳癌成對檢體之mRNA表現--------------------49
圖八：自動基因定序結果圖形----------------------------50
圖九：臨床乳癌成對檢體之蛋白質表現---------------------51
圖十：免疫組織染色結果-------------------------------52
圖十一：免疫組織染色結果-----------------------------53
圖十二：Real-time quantitative PCR作腫瘤與非腫瘤乳房組織
Genomic DNA copy number的比較-----------------54
表一：乳癌檢體分類及免疫組織染色之結果-----------------55
表二：14-3-3 gamma表現與病人乳房腫瘤病理關連性----------56
圖十三：Real-time quantitative PCR原理--------------------57

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