尿路上皮癌之尿液與組織蛋白質體研究

Studies on Urinary and Tissue Proteome of Urothelial Carcinoma

盧誌明(Chih-Ming Lu)

指導教授 ： 葛應欽

共同指導教授 ： 黃俊雄(Chun-Hsiung Huang)

高雄醫學大學/醫學院/醫學研究所/博士(2011年)

https://doi.org/10.6832/KMU.2011.00005

摘要

研究背景　尿路上皮癌是指發生於膀胱、尿道、輸尿管、腎盂及腎盞移形上皮的癌症。台灣地區尿路上皮癌有增加趨勢，五年間尿路上皮癌發生率至少增加四分之一，女性尿路上皮癌發生率增加的速度比男性快。上段尿路上皮癌有易復發、轉移、需終身追蹤的傾向，所以希冀應用此一技術，尋找上段尿路上皮癌的可能腫瘤標記。尿路上皮癌的症狀以血尿為主，但是其他泌尿道疾病也以血尿表現。由於現有的膀胱癌腫瘤標記、細胞學檢查都無法取代有顯影劑過敏危險的靜注腎盂攝影檢查、以及侵入性膀胱鏡檢查引起的不舒服感，所以找尋診斷正確率高的腫瘤標記是很重要任務。目前研究腫瘤標記的新技術中，蛋白質體研究被認為是極具潛力的一項工具。本研究計畫以二維凝膠電泳，發現有效的尿路上皮癌腫瘤指標，並且提出腫瘤指標的簡易檢驗方法。因為低含量蛋白質在二維凝膠電泳圖譜上常常被白蛋白和免疫球蛋白等豐富性蛋白質遮掩，而不易被發現。本研究首先要發展新的分層法，利用不同的鹽類濃度將豐富性蛋白質移除，並發現更多的低含量蛋白質。研究者採用弱陰離子交換色層分析技術來達到此一目的。本研究期待比較上段尿路上皮癌患者與健康者的尿液，找出差異蛋白質，做為候選的腫瘤標記。
受試者與研究方法　在第一個研究納入二十位健康者的尿液，採用 DEAE-Sephacel 樹脂做為非定量沖洗液逐步弱陰離子交換色層分析的交換樹脂，以液態色層分析串聯質譜 (LC-MS/MS) 確定蛋白質身份。第二個研究除這二十位健康者的尿液之外，增加十三位上段尿路上皮癌患者的尿液，並收集其中三位的癌組織與正常尿路上皮組織。研究者先以非定量沖洗液逐步弱陰離子交換色層分析的交換樹脂分層兩組的混合尿液 (pooled urine sample)，再以 LC-MS/MS 及套裝電腦程式找出兩組的差異蛋白質，研究者繼續針對其中數種差異蛋白質進行個別人的尿液進行西方墨跡分析。在本研究最後，研究者以西方墨跡分析比較上段尿路上皮癌患者的癌組織與正常尿路上皮組織，確定差異蛋白質的來源。第三個研究收集十二位腎盂尿路上皮癌患者的癌組織與正常尿路上皮組織，以西方墨跡分析比較候選的腫瘤標記在兩者的表現。第四個研究則收集七十八位原發性非肌肉侵犯性膀胱尿路上皮癌患者的組織、無復發存活時間、期別、級別、抽菸習慣等因子，以逆轉錄聚合酶鏈反應 (RT-PCR) 分析可誘發性一氧化氮合成(酉每)的訊息核糖核酸表現，以了解可誘發性一氧化氮合成(酉每)的訊息核糖核酸表現與膀胱癌復發的關聯性。
結果　採用 DEAE-Sephacel 樹脂做為非定量沖洗液逐步弱陰離子 (0 M、50m M、100 mM、1 M NaCl) 交換色層分析的交換樹脂可以將尿液蛋白分為四層 (Fraction Unbound、NaCl-1、NaCl-2、NaCl-3)，大多數豐富性蛋白質會出現在 Fraction Unbound，有更多的非豐富性蛋白質可被液態色層分析 - 質譜分析 /質譜分析 (LC-MS/MS) 確定蛋白質身份。第二個研究應用此一技術，發現上段尿路上皮癌患者與健康者的尿液的二維圖譜各有1,028 及 608 個蛋白質點。本研究發現有 49 個差異蛋白質。其中三個zn-alpha-2-glycoprotein (ZAG) 、 calreticulin (CRT) 、 haptoglobin (Hp) 進行個別上段尿路上皮癌患者與健康者的尿液西方墨跡分析，發現三者在上段尿路上皮癌患者尿液的表現高於健康者尿液。 CRT 被用來進行上段尿路上皮癌患者的癌組織與正常尿路上皮組織的西方墨跡分析，發現在上段尿路上皮癌組織的表現高於患者的正常組織。第三個研究增加受試者至 12 位，發現 CRT 、 Rho GDI 、 Hsp70 、 PDX2大多數在腎盂尿路上皮癌組織的表現高於患者自身的正常組織。第四個研究最後有七十一位原發性非肌肉侵犯性膀胱尿路上皮癌患者可以完成研究，未復發組的追蹤時間中位數是 34 個月，復發組的追蹤時間中位數是 12 個月，可誘發性一氧化氮合成(酉每)的訊息核糖核酸表現為陽性者有較高的復發率 (18.7% vs. 2.6%, p=0.04) ，而且無復發存活曲線分析也達到統計顯著 (p=0.019)。
結論　本研究發展一個有效發現低豐富蛋白質的非定量沖洗液逐步弱陰離子交換色層分析法，找到 49 個尿路上皮癌的差液蛋白質，發現 ZAG 、 Hp 、 CRT 、 Rho GDI 、 Hsp70 、 PDX2　是尿路上皮癌的可能腫瘤標記。此外，　iNOS mRNA 的表現是膀胱癌復發的預測因子。

關鍵字

蛋白質體 ； 尿路上皮癌 ； 弱陰離子交換色析法 ； 尿液

並列摘要

Background: Low-abundance proteins are difficultly observed on the two-dimensional gel electrophoresis (2-DE) maps of urine proteome, because they are usually obscured by high-abundance proteins such as albumin and immunoglobulin. In this study, a novel fractionation method was developed for enriching low-abundance proteins by removing high-abundance proteins and progressive elution with salts of various concentrations. We applied the WAX technique to find out tumor markers. Because upper urinary tract urothelial carcinoma (UTUC) has a propensity of recurrence and migration and worse prognosis, life-long surveillance is deemed necessary. Tumor biomarkers are desirable for clinicians and patients. Therefore, urine from patients withUTUC was collected to analyze the different proteins compared with healthy individuals’ urine. In addition, we conducted a study to investigate the association between the positive iNOS mRNA expression and recurrence of bladder cancer.
Subject and Methods: In the present study, samples from 13 UTUC patients and 20 healthy controls were examined by urine proteomic analysis and western blot analysis. A stepwise weak anion exchange chromatography with DEAE-Sephacel resin and non-fixed volume elution was used to fractionate urine proteome before performed two-dimensional gel electrophoresis (2-DE). In the iNOS study, 71 patients with primary non-muscle-invasive bladder cancer were enrolled in this study. Tumor tissues were harvested during transurethral resection of bladder tumors. All operations were performed at the same hospital. Recurrence-free patients were followed up for at least 1 year unless tumors recurred during that time.
Results: Stepwise weak anion exchange (WAX) chromatography, which applied DEAE-Sephacel resin with non-fixed volume elution, was used to fractionate urine proteome prior to performing 2-DE. Urine proteome was separated into four fractions by progressively eluting the column with 0M, 50mM, 100mM, and 1M NaCl solutions. Most of the heavy and light immunoglobulin chains appeared in the eluent. After the high-abundance proteins were removed, various low-abundance proteins were enriched and could be easily identified. The potential of this method for obtaining diversified fractionations was demonstrated by eluting the column separately with Na2SO4 and MgCl2 solutions. The 2-DE maps of the fractions eluted with these different salt solutions of identical ionic strength revealed markedly different stain patterns. Using these procedures, the high-abundance proteins were filtered and the low-abundance proteins were identified. On the 2-D maps, 1,028 and 608 proteins spots were identified from the samples of the UTUC group and the healthy control group respectively. UTUC group’s 49 differential protein spots were distinguished from healthy controls. Western blot analysis on the urine samples was carried out for three potential UTUC markers, i.e. zn-alpha-2-glycoprotein (ZAG), calreticulin (CRT), and haptoglobin (Hp). All three candidate proteins demonstrated higher expression in UTUC patients than in healthy controls. Furthermore, CRT appeared higher expression in tumor tissues than that of the normal epithelium from UTUC patients. In the iNOS study, The median intervals of follow-up were 34 months in the recurrence-free group and 12 months in the recurrence group. Inducible NOS mRNA was detected using the RT-PCR-based method. Bladder cancer patients with positive iNOS mRNA expression had a higher recurrence risks (18.7% vs. 2.6%, p=0.04). After adjusting for other risk factors, a statistical significance remained to be reached (p=0.04, hazards ratio=13.27, 95% CI=1.07-164.36). The patients also had reduced recurrence-free survival (p=0.019).
Conclusion: The present study demonstrated that this fractionation method could be applied for purposes of enriching low-abundance proteins and obtaining diversified fractionations of urine, and potentially other proteomes. We propose ZAG, CRT, and Hp as potential tumor biomarkers for UTUC diagnosis. Overexpression of iNOS mRNA may be a useful prognostic indicator of bladder cancer.

並列關鍵字

proteome ； urothelial carcinoma ； weak anion exchange chromatograpgy ； urinary

參考文獻

Adachi, J., Kumar, C., Zhang, Y., Olsen, J.V. & Mann, M. (2006) The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol, 7, R80.

Aebersold, R. & Mann, M. (2003) Mass spectrometry-based proteomics. Nature, 422, 198-207.

Anderson, N.G., Anderson, N.L. & Tollaksen, S.L. (1979) Proteins of human urine. I. Concentration and analysis by two=dimensional electrophoresis. Clin Chem, 25, 1199-1210.

Aviles-Santa, L., Alpern, R. & Raskin, P. (2002) Reversible acute renal failure and nephritic syndrome in a Type I diabetic patient. J. Diabetes Complication, 16, 249-254.

Awadallah, S. & Atoum, M. (2004) Haptoglobin polymorphism in breast cancer patients form Jordan. Clin Chim Acta, 341, 17-21.