大鼠腎臟中糖尿病相關蛋白之蛋白質體之螢光高效能液相層析串聯質譜分析

根據美國腎臟病資料登錄系統在2007年統計的數據，台灣的末期腎病變發生率或盛行率皆為世界第一，其中因糖尿病所導致的比例高達43.1%。本研究中，建立腎臟之蛋白質體分析方法，以偵測在糖尿病腎病變初期，即發生表現量改變的蛋白質，以利於診斷或治療。利用腹腔注射streptozotocin 80 mg/kg body weight 誘導七週大的Sprague-Dawle大鼠成糖尿病鼠，控制組則注射等量檸檬酸緩衝液，在誘導後1週、4週、12週、24週等四個不同的時間點分別犧牲，取約100毫克右腎皮質將之均質後定量，並利用螢光衍生化試劑DAABD-Cl與其反應後，以螢光高效能液相層析儀分離，將糖尿病狀態下表現量產生差異的蛋白質收集後，經由蛋白酶消化並以液相層析串聯質譜儀(LC-MS/MS)定性此蛋白質。實驗結果顯示，誘導4天後，大鼠的血糖值皆高於300 mg/dL，其飲食、排泄、毛色等均顯示大鼠已經成功被誘導成糖尿病鼠。大鼠的微白蛋白尿在誘導後24週才有統計上的差異。然而蛋白質體的分析結果顯示，誘導後1、4、12、24 週的大鼠分別有7、1、9、18根peak有統計上的差異(p<0.05)，顯示蛋白質的變化早於微白蛋白尿。本研究結果將有助於糖尿病腎病變的早期診斷及新藥的開發。

關鍵字

糖尿病腎病變；蛋白質體；螢光高效能液相層析串聯質譜；螢光衍生化試劑DAABD-Cl

並列摘要

Diabetic nephropathy (DN) is a complication of diabetes mellitus which may lead to end-stage renal disease. Throughout its early course, DN has no symptoms. The first laboratory abnormality is a positive sign by microalbuminuria test. However, at this stage, a kidney biopsy clearly shows DN. Therefore, this study is designed to establish a new method to identify the differential expression of proteins in the kidney of control and streptozotocin (STZ)-induced diabetic rats in order to discover specific indicators for early diagnosis of DN. The Spregue-Dawley rats were sacrificed at 1, 4, 12, 24 weeks after i.p. injection of sodium citrate buffer or STZ. A thiol-specific fluorogenic reagent, 4-[2-(dimethylamino)ethyl]-7-chloro-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) was utilized for the derivatization of proteins and then the derivatives were quantified and collected by high performance lipid chromatography with fluorescence detection (FD-HPLC). The isolated derivatives will be further identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with the MASCOT database searching system. After 1 week of injection, the expressions of approximately 7 proteins significantly changed. Only one protein had significantly changed after 4 weeks of injection. However, after 12 weeks of treatment, the number of altered proteins rose to 9. In addition, the changes appeared before the increase of microalbumin in the urine. The results may serve to find new biomarkers to improve early detection and new drug development in diabetic-related nephropathy.

並列關鍵字

DAABD-Cl ； Proteome ； FD-LC-MS/MS ； Diabetic nephropathy

參考文獻

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大鼠腎臟中糖尿病相關蛋白之蛋白質體之螢光高效能液相層析串聯質譜分析

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