馬兜鈴酸誘導小鼠腎炎中蛋白質體之螢光高效能液相層析串聯質譜分析

Proteome analysis of aristolochic acid nephropathy in mouse kidney by fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method

張文歆(Wen-Shin Chang)

指導教授 ： 陳世銘 副教授

臺北醫學大學/藥學院/藥學研究所/碩士(2011年)

摘要

馬兜鈴酸腎病變 (aristolochic acid, AAN) 是一種快速進展到末期腎臟疾病的間質性腎炎，其原因是誤用含有馬兜鈴酸的生藥，而目前唯一的確診方式為腎臟組織切片。本研究利用蛋白質體分析方法，以鑑定在馬兜鈴酸腎病變後表現量改變的蛋白質，作為疾病特定的指標。利用投予AA飲用水 0.5 毫克/公斤 體重/天 ，誘導6週齡雄性C3H/He小鼠8週，於誘導後8週、10週及12週不同時間點分別犧牲。取得腎臟組織經均質後，利用螢光衍生化試劑DAABD-Cl與其反應，以螢光高效能液相層析儀 (FD-HPLC) 分離並定量，收集表現量有差異的蛋白質以液相層析串聯質譜儀 (LC-MS/MS) 與Mascot數據庫檢索系統定性。實驗結果顯示，誘導8週後，AA組小鼠的血糖值、血中尿素氮、尿蛋白及尿中N-acetyl-β-D-glucosaminidase (NAG) 均有上升；腎組織鏡檢發現腎小管萎縮、間質細胞浸潤及纖維化。在誘導後8週、10週及12週的AA組小鼠，與正常組小鼠比較，分別有16、54、24根波峰有顯著的差異 (p < 0.05)，蛋白質經鑑定為與抗氧化、細胞外基質蛋白質與其相關訊息路徑、發炎、細胞凋亡及ATP合成有關。本研究結果將有助於找到馬兜鈴酸腎病變的生物標誌，以應用至早期診斷，並更加了解馬兜鈴酸腎病變的病理機轉。

關鍵字

馬兜鈴酸腎病變 ； 蛋白質體 ； 螢光高效能液相層析串聯質譜

並列摘要

Aristolochic acid nephropathy (AAN) is a progressive interstitial nephritis which rapidly leading to end-stage renal disease. The reason is that people misuse the herb containing AA. So far, using kidney biopsy is the only way to diagnose. Therefore, this study was designed to establish a new method of proteomics to identify the differential expression of proteins in AAN in order to find out the specific indicators of AAN. The 6 week-old male C3H/He mice were treated with AA (0.5 mg/kg/day) for 8 weeks. The mice were sacrificed at 8, 10 and 12 weeks since the treatment began. The homogenate of the kidney was derivatized with DAABD-Cl. The dirivatized proteins were separated and quantified by high performance liquid chromatography with fluorescence detection (FD-HPLC). The differential proteins were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with MASCOT database searching system. After 8 weeks of AA administration, they were showed renal injury by increasing blood glucose, blood urea nitrogen (BUN), urine protein and N-acetyl-β-D-glucosaminidase (NAG). In addition, tubular atrophy, interstitial infiltration and fibrosis were found in histopathology. The significantly altered expression between the normal and AA group at 8, 10 and 12 weeks were 16, 54 and 24 peaks, respectively. Those proteins were identified as anti-oxidant, extracellular matrix related, inflammatory, apoptotic and in ATP synthesis. This protein profile may serve to find new biomarkers to improve early detection and realize more the mechanism of AAN.

並列關鍵字

Aristolochic acid nephropathy (AAN) ； proteomics ； fluorogenic derivatization-liquid chromatography-tandem mass spectrometry

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