順氯氨鉑引發小鼠腎臟損傷下的蛋白質體之螢光高效能液相層析串聯質譜分析

Proteome analysis of altered proteins in cisplatin induced mouse acute kidney injury (AKI) using fluorogenic derivatization–liquid chromatography–tandem mass spectrometry (FD-LC-MS/MS) method

陳崇慧(Tsung-Hui Chen)

指導教授 ： 李仁愛

臺北醫學大學/藥學院/藥學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00060

摘要

順氯氨鉑為一被廣泛使用和具有高效用的化療藥物，現已證明其可應用在治療睪丸，卵巢，膀胱和胃腸道等癌症，其療效與其投與劑量相關，然而給予劑量愈高也愈容易引發腎毒性，約有三分之一患者在投與此藥後發生腎損傷，其臨床使用因而受限。本研究的目的是探討經順氯氨鉑引發腎病變後產生差異的蛋白質，以助於早期診斷、預防和分類順氯氨鉑引發之急性腎損傷。6週齡雌性BALB / c小鼠腹腔注射順氯氨鉑5毫克/每公斤體重/天，連續給藥3天或5天後犧牲取得尿液、血清及腎組織。由血中尿素氮、尿中尿酶NAG及腎臟組織確認小鼠腎損傷被成功誘導。將腎均質液以DAABD - Cl衍生化後，利用螢光高效液相層析法分析。最後，將表現量產生變化的蛋白質經LC-MS/MS與 Mascot數據庫檢索系統分析，得到結果。連續給予順氯氨鉑3天的小鼠，與正常小鼠的腎臟相較，共有Catalase、Parkinson disease protein7…等，33個蛋白質的表現出現顯著差異 (P<0.05)；連續給藥5天的小鼠腎臟組織中，僅有glial fibrillary acidic protein、peroxisomal membrane protein 20 …等，9個蛋白質的表現有顯著變化 (P<0.05)。由此結果可知，順氯氨鉑可能經由抗氧化和發炎機制誘導小鼠發生急性腎損傷；並且在5天之後，可能因損傷過於嚴重使得蛋白表現趨於鈍化。本研究成功建立以螢光高效液相層析分離小鼠腎臟蛋白質，它可能有助於找到新的生物標誌物，以提高早期發現順氯氨鉑引起的急性腎損傷。

關鍵字

順氯氨鉑 ； 腎毒性 ； 蛋白質體學

並列摘要

Cisplatin, one of the most widely used and most potent chemotherapy drugs, has been proven its clinical efficacy on testicular, ovarian, bladder and gastrointestinal cancer etc., but its renal toxicity which may lead to acute kidney injury (AKI) has limited the utility. The prevalence of cisplatin renal toxicity is high, occurring in about one-third of patient after cisplatin treatment. The purpose of this study was to find out the altered proteins in cisplatin nephropathy, with a view to prevent, diagnose and classify acute kidney injury effectively. The 6-week-old female BALB/c mice were intraperitoneally administered cisplatin at a level of 5 mg/kg/day for 3 or 5 days to induce acute kidney injury successfully. The homogenate of the kidney was derivatized with 4- [2-(dimethylamino)ethylaminosulfonyl]-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl), and subjected to proteome analysis by Fluorogenic Derivatization- High-Performance Liquid Chromatography (FD-HPLC). Finally, the altered proteins were identified by LC-MS/MS with MASCOT database searching system. According to N-acetyl-β-D-glucosaminidase (NAG) and serum blood urea nitrogen (BUN), the renal function had already been disrupted after 3 days or 5 days of cisplatin injection. The results showed whether 3 days or 5 days of cisplatin injection, both of their proteins changed significantly. Between the normal and AKI tissues of the 3-day treatment group, more than 33 proteins has significantly different expressions (p<0.05). However, only 9 proteins has significantly different expressions (p<0.05) in the 5-day treatment group. It may be derived from the severe damage of kidney, and leads to renal cell death. In the present study, a highly effective method for analyzing the altered proteins after cisplatin treatment was developed. The identification of the altered proteins will be presented. It may serve to find new biomarkers to improve early detection and new drug development in cisplatin induced AKI.

並列關鍵字

cisplatin ； nephrotoxicity ； proteomics ； DAABD-Cl ； FD-LC-MS/MS

參考文獻

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