在癌細胞中經常觀察到腫瘤抑制基因或蛋白的缺失，並伴隨其他基因或蛋白質調控情形的改變。因此，在本篇實驗中，我們利用siRNA的方式在正常乳腺細胞 (ME16C)降低一種乳癌中常見缺失的腫瘤抑制蛋白質—PTEN，並藉由二維差異電泳及基質輔助雷射-飛行時間式質譜儀分析蛋白質體的改變，鑑定出十個在PTEN缺失時具差異表現的蛋白質。分析發現，它們大部分具有致癌或腫瘤抑制的能力，例如調控細胞生長 (alpha-enolase)、增生 (NF2)、細胞骨架重組 (Hsp27)、凋亡 (ST20，suppressor of tumorigenicity 20)等功能，其中也有未知功能的蛋白質 (CCDC90A)。我們更進一步的利用西方墨點法和即時定量聚合酶連鎖反應，來觀察這些蛋白質是在基因轉錄或蛋白質轉譯層級受到PTEN表現量改變的調控；最後比較這些蛋白質在正常乳腺細胞 (MCF10A)、低侵略性 (MCF7)和高侵略性 (MDA231)乳癌細胞中，基因及蛋白質的表現情形。綜合分析之後，我們提出這些蛋白質可能受PTEN調控的機制及其在乳癌進程中的意義和關聯性；其中CCDC90A這個未知功能的蛋白質更首次被我們發現受PTEN調控，且在模式細胞株中具有隨癌化程度增加表現的趨勢。期待透過本篇研究，對於往後瞭解這些蛋白質受調控的機制及其與癌症的關係具有新的方向及幫助。

Loss of tumor suppressor is a common phenomenon in tumor cell. The defect accompanies with alterations of gene transcription and protein expression profiles. In this study, a frequently lost tumor suppressor in breast cancer, PTEN, is knockdown by siRNA in human mammary cell. Protein profile of the PTEN depleted cells was analyzed by two-dimension differential gel electrophoresis combined with matrix-assisted laser desorption ionization/time of flight mass spectrometry. Ten of the protein spots were identified, and most of them play roles in oncogenesis or tumor suppression. These proteins participate in cell growth (alpha-enolase), proliferation (NF2), cytoskeleton rearrangement (Hsp27), and apoptosis (suppressor of tumorigenicity 20, ST20). Additionally, a functional unknown protein, CCDC90A, is also found. We subsequently used Western blotting and real-time quantitative polymerase chain reaction to examine whether the differential expressions are regulated at gene transcriptional or protein translational level. The gene expression and quantity of the identified proteins were compared among normal mammary cell (MCF10A), non-invasive and invasive breast cancer cell (MCF7 and MDA231, respectively). In summary, we investigated the proteins that differentially expressed in PTEN knock-down cells and analyzed the correlations to breast tumor progression. We identified that CCDC90A is negatively regulated by PTEN expression and up-regulated through tumor progression; a phenomenon that has not been reported. This study offers extended information for future research on those proteins to explore the occurrence and progression of breast cancer.

緒論
材料與方法
1化學藥品來源
2-1細胞株及其培養條件
2-2-2細胞繼代
2-2-3冷凍保存細胞
2-2-4解凍細胞
3藉轉染短干擾核糖核酸降低蛋白質表現
4藉轉染質體增加蛋白質表現
5萃取細胞蛋白質
5-1二維電泳之蛋白質萃取
5-2西方墨點法之蛋白質樣品萃取
5-3蛋白質濃度定量
6二維差異電泳 (2D-DIGE)
6-1蛋白質螢光標定處理 (CyDye labeling)
6-2第一維等電點電泳— isoelectric focusing (IEF)
6-3第二維電泳 (SDS-PAGE)
7蛋白質膠內酵素水解(In-gel digestion)
8基質輔助雷射脫附游離飛行式質譜儀
9西方墨點法 (Western blot)
10核糖核酸萃取 (RNA extraction)
11反轉錄聚合酶連鎖反應(Reverse Transcription PCR)
12聚合酶連鎖反應 (polymerase chain reaction)
13即時定量聚合酶連鎖反應
附表
實驗結果
1.分析ME16C正常乳腺細胞缺乏PTEN時蛋白質體表現改變情形
2.表現量差異的蛋白質鑑定
3.蛋白質位置和功能性分類
4.利用西方墨點法再次確認鑑定出的蛋白質
5.利用即時定量聚合酶連鎖反應確認鑑定出的蛋白質是否受到基因轉錄層級的調控
6.利用西方墨點法確認鑑定出的蛋白質在正常乳腺細胞、低侵略性和高侵略性乳癌細胞中表現情形
7.利用即時定量聚合酶連鎖反應確認鑑定出的蛋白質在正常乳腺細胞、低侵略性乳癌細胞和高侵略性乳癌細胞中是否受到基因轉錄層級的調控
討論
參考資料
圖表

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