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系統識別號 U0007-0608201109385000
論文名稱(中文) 奈米探針輔助親和性質譜法偵測血清攝護腺特定抗原- 可為攝護腺癌研究的平臺
論文名稱(英文) Detect Serum Prostate Specific Antigen by Nanoprobe-Based Affinity Mass Spectrometry - A Novel Prostate Cancer Research Platform
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 99
學期 2
出版年 100
研究生(中文) 溫玉清
研究生(英文) Yu-Ching Wen
學號 M118098002
學位類別 碩士
語文別 英文
口試日期 2011-07-01
論文頁數 50頁
口試委員 委員-稅皓靄
委員-華國媛
委員-鄭朝文
共同指導教授-李良明
指導教授-陳振漢
中文關鍵字 攝護腺癌  攝護腺特定抗原  奈米探針輔助親和性質譜法  基質輔助雷射脫附/游離飛行時間質譜儀  攝護腺癌細胞株 
英文關鍵字 prostate cancer  prostate specific antigen  nanoprobe-based affinity mass spectrometry  matrix-assisted laser desorption/ionization time-of-flight mass spectrometry  LNCaP 
學科別分類
中文摘要 攝護腺(前列腺)癌是美國最常見的男性惡性腫瘤。據衛生署統計,二○○九年台灣國人男性攝護腺癌的發生率排名於男性癌症第五位。近五年國內每年有超過千位攝護腺癌新病例被診斷出來,死亡率的排名於男性癌症死亡十大疾病為第七位。近年來由於醫學進步,經由早期的診斷及各種有效的治療方法,可提升攝護腺癌的存活率。血清攝護腺特定抗原(serum prostate specific antigen,簡稱PSA)檢查是目前公認最佳早期偵測攝護腺癌的診斷工具之一。當血清中的攝護腺特定抗原濃度超過4毫微克/毫升時,即可視為攝護腺癌的高度危險群。但PSA值上升的原因不只有攝護腺癌,其他最常見的原因有攝護腺肥大或是攝護腺炎。因而需經由攝護腺切片來確定,但並非全無風險。所以,如何減少不必要的攝護腺切片為一重要課題。
在本篇論文中,我們應用在之前所開發的奈米探針輔助親和性質譜法(nanoprobe-based affinity mass spectrometry, NBAMS)做為攝護腺特定抗原的免疫測定方法。利用表面結合有攝護腺特定抗原抗體的磁性奈米探針,藉由抗體對抗原的高專一性作用力,再結合具有高靈敏度的基質輔助雷射脫附/游離飛行時間質譜儀針對含量極低且不易游離的高度醣化攝護腺特定抗原樣品進行偵測。以此分析方法可以在人體血清中,偵測低至五毫微克/毫升的標準儲精囊攝護腺特定抗原。並同時在實驗室培養對男性睪固酮依賴性及非依賴性的兩株攝護腺癌細胞,運用此方法亦可精確取得其分泌之攝護腺特定抗原,且在基質輔助雷射脫附/游離飛行時間質譜儀中,發現其在某些特定訊號中和標準品攝護腺特定抗原有所不同。此平台的建立可在早期診斷攝護腺癌中有所進展,甚至未來可得知攝護腺癌對男性荷爾蒙阻斷治療失效的潛在性以改善攝護腺癌個人化治療。然而,後續還需更多的血液標本來做驗證。
英文摘要 Prostate Cancer is the most common male malignant tumor in U.S.A.. According to the statistics of Department of Health, ROC in 2009, the prostate cancer is the 5th common cancer of male in Taiwan. In recent 5 years, there are more than one thousand new cases diagnosed and the mortality rate ranks 7 among the top 10 cancers. However, because of the improvement of medical managements, through early diagnoses and other effective treatments, the survival rate is upgraded. The PSA measurement is one of the most effective ways to detect prostate cancer. When the prostate specific antigen level in serum is over 4ng/ml, it can be regarded as the high risk sign of prostate cancer. However, in addition to the prostate cancer, other reasons such as benign prostate hyperplasia and prostatitis can also contribute to the increase of PSA level. Therefore, the prostatic biopsy is necessary, but with risk exists. Hence, how to decrease the unnecessary prostatic biopsy by screening the specific biomarker for clinical practice is an important issue.
In this study, we apply nanoprobe-based affinity mass spectrometry (NBAMS) as the method of immunoassay for PSA from human serum. Combination of anti-free PSA antibody-conjugated magnetic nanoparticles (anti-PSA@MNPs), and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, we can enrich and detect high glycosylative PSA from human seminal plasma. After a series optimization procedures, human serum PSA and the PSA from cultured prostate cancer cell lines can be detected level around 5 ng/ml. With this NBAMS, we can get the PSA accurately and find the different significant signals between prostate cancer cell lines and standard seminal specimen of PSA. Take together; the platform established in this study demonstrated that it can use to advance the diagnosis of early stage prostate cancer as well as the hormone refractory potential of the disease to improve the personal therapy of the prostate cancer in future. However, more samples for further validation are necessary.
論文目次 中文摘要…………………………………………………………… i
Abstract…………………………………………………………… iii
1. Introduction………………………………………………… 1
1. Prostate cancer and Biomarker……………………… … 1
1-2. Prostate Specific Antigen…………………………… 2
1-3. Nanoprobe-based affinity mass spectrometry……… 4
1-4. Purpose……………………………………………………… 5
2. Materials and methods……………………………………… 7
2-1. Preparation of Antibody-Conjugated Magnetic Nanoparticles (Ab@MNP)……………………………………… 7
2-2. Human Serum Samples and ELISA Measurement………… 7
2-3. Instrumtion………………………………………………… 8
2-4. Prostate cancer cell culture………………………… 8
2-5. Immunoaffinity Extraction by Ab@MNP……………… 9
2-6. Experimental Design and Workflow……………………… 10
3. Results…………………………………………………………… 11
3-1.1 Seminal free PSA identification.……………………… 11
3-1.2 Anti-PSA@MNPs background……………………………… 11
3-2. Enrichment Specificity…………………………………… 12
3-3. The optimization of anti-PSA@MNPs count…………… 13
3-4. Profiling of Serum Proteins……………………………… 14
3-5. Evaluation of Detection Sensitivity…………………… 15
3-6. Compare the detection of the limitation of anti-PSA@MNPs by SDS-PAGE analysis………………………………… 16
3-7. Detect PSA from prostate cancer cell lines……………16
3-8. Enrichment of PSA from patient’s serum……………… 17
4. Discussion…………………………………………………………19
5. Conclusion and Perspective……………………………………28
6. References…………………………………………………………30
7. Figures ……………………………………………………………37
8. Table……………………………………………………………… 50
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