The effects of organic and inorganic selenium compounds on oxidative stress and proteomic expression in aged mice brain.

目錄 i 圖目錄 v 表目錄 vii 致謝 viii 縮寫對照表 x 中文摘要 xi Abstract xiii 第一章 緒論 1 第一節 前言 1 第二節 文獻回顧 2 一、 老化對腦部與神經系統之影響 2 1. 老化對腦部之影響 2 2. 老化對神經傳導系統之影響 2 二、 老化對抗氧化防禦系統之影響 5 1. 抗氧化防禦系統與老化之影響 6 三、 老化對行為能力之影響 11 四、 硒之介紹 12 1. 硒之介紹 12 2. 硒之建議量、缺乏症及硒中毒 12 3. 硒之化學形式 16 4. 硒的消化、吸收及代謝 21 5. 硒之功效 23 五、 老化對腦部蛋白質體之影響 24 1. 蛋白質體學 (proteomics) 之定義 24 2. 蛋白質體學之研究工具 25 3. 蛋白質體學在腦部之研究 26 第三節 研究目的與設計 28 一、 研究目的 28 二、 研究設計 29 三、 實驗設計說明 30 第二章 材料與方法 31 一、 動物飼養 31 二、 飼料之製備 31 三、 動物犧牲 33 四、 血樣收集 34 五、 組織均質 34 六、 蛋白質測定 34 七、 免疫轉印法 36 1. SDS膠體電泳 36 2. 蛋白質免疫轉印法 40 3. 酵素免疫染色法 42 4. 膠體染色 43 5. 膠體乾片製作 44 八、 蛋白質體學分析 45 1. 腦組織蛋白質萃取與收集 45 2. 蛋白質回溶 46 3. 蛋白質測定 (2-D Quant kit) 47 4. 覆水作用 (rehydration) 48 5. 第一維：等電點聚焦 (IEF) 48 6. 平衡作用 (equilibration) 49 7. 第二維：SDS膠體電泳 50 8. 膠體染色 52 9. 膠體影像處理與比對 54 10. 膠片保存 55 九、 組織中MAO活性之測定 55 十、 血漿中ORAC總抗氧化能力測定 57 十一、 組織中TBARS含量之測定 58 十二、 組織中Carbonyl含量之測定 59 十三、 組織中Catalase活性之測定 62 十四、 組織中GPx活性之測定 63 十五、 組織中SOD活性之測定 64 十六、 組織中維生素C含量之測定 65 十七、 組織中GSH含量之測定 67 十八、 統計 68 第三章 結果 70 一、 動物生長與攝食情形 70 二、 組織重量 70 三、 腦部與肝臟MAO酵素活性之比較 71 四、 腦部MAO-B蛋白質表現量之分析 72 五、 血漿總抗氧化能力之比較 72 六、 腦部與肝臟脂質過氧化程度之比較 72 七、 腦部與肝臟蛋白質氧化程度之比較 73 八、 腦部與肝臟酵素性抗氧化系統之比較 73 九、 腦部與肝臟非酵素性抗氧化系統之比較 74 十、 腦部蛋白質體之分析 75 第四章 討論 96 一、 硒補充對動物生長與組織重之影響 96 二、 硒補充對老齡小鼠體內氧化壓力之影響 97 三、 硒補充改善老齡小鼠體內抗氧化防禦系統之作用 102 四、 硒補充對腦部蛋白質體之影響 106 第五章 總結與未來展望 110 一、 總結 110 二、 未來展望 110 參考文獻 111[[abstract]]硒 (selenium, Se) 為人體所必需之微量營養素。眾多實驗證實，硒具有抗氧化與預防老化之功效。且硒的抗氧化作用是藉由硒化合物之氧化還原反應，防禦過氧化物之作用來達成。硒具有兩種不同型態：無機硒 (inorganic selenium) 與有機硒 (organic selenium)，皆具有抗氧化功效。而生物體的老化是種錯綜複雜之現象，均會受到自由基 (free radicals) 和氧化壓力 (oxidative stress) 等影響。研究顯示，老化期間腦部抗氧化防禦系統均遭受改變，文獻指出硒的補充可降低腦部因老化所致之氧化傷害。因此，本研究目的為不同硒形式的補充，是否可預防老齡小鼠腦部因老化之相關氧化損傷和利用二維電泳探討蛋白質體表現量之影響。本研究採用69-73週齡BALB/c雌鼠，依體重隨機分組，共分3組。分別餵食不同實驗飼料分成：(1) 老齡控制組 (older control簡稱OC)、(2) 無機硒組 (selenite簡稱SE；硒當量含0.5 mg/kg diet)、(3) 有機硒組 (selenium yeast簡稱SY；硒當量含0.5 mg/kg diet)；另有一組10週齡之(4) 年輕控制組 (young control簡稱YC)，分別餵食14週後犧牲取血漿、腦部及肝臟。結果顯示，OC組腦部MAO-B酵素活性明顯高於YC組，給予硒補充則有抑制其含量，而肝臟MAO-B酵素活性則無差異。腦部MAO-B蛋白質表現量4組間無顯著差異。老化會改變血漿總抗氧化能力，硒補充則可回復至與YC組相同。腦部與肝臟中脂質過氧化與蛋白質氧化之含量，也會因老化而顯著增加，硒補充後則顯著降低。體內酵素性抗氧化防禦系統 (包含SOD、Catalase和GPx) 與非酵素性抗氧化防禦系統 (維生素C和GSH) 也會因老化而有所改變，給予硒補充則有改善因老化所影響的酵素性抗氧化系統 (包含SOD、Catalase和GPx) 與非酵素性抗氧化系統 (維生素C和GSH) 之狀態。二維電泳分析YC組和OC組可明顯觀察到18個具有差異蛋白質點，給予老齡有機硒與無機硒補充後也分別各有12個與18個差異蛋白質點，其中，OC組圖譜上之A與C蛋白質點因老化後蛋白質量大於YC組，而B與D蛋白質點因老化後蛋白質量小於YC組，且A蛋白質點在老齡硒補充 (SE與SY組) 後均使蛋白質量減少，B蛋白質點在老齡無機硒補充 (SE組) 後使蛋白質量增加，C蛋白質點在老齡硒有機硒補充 (SY組) 後使蛋白質減少，D蛋白質點在老齡有機硒補充 (SY組) 後使蛋白質增加，可見硒的補充均使這些蛋白質表現量回復至與YC組相當。未來將進一部分析確認差異蛋白質之組成。本實驗結果證實，老化會增加氧化損傷的程度，給予不同硒型式補充具有預防之功效。老化與不同硒型式補充均造成腦部蛋白質體的改變，雖然現在並未知是什麼蛋白質發生變化，但未來還需鑑定這些蛋白質體，探討這些蛋白質體之間對腦部相關機制之影響。 Selenium is a dietary essential trace element for human’s health and an integral part of many proteins with catalytic and structural functions. Se exists naturally in inorganic and organic forms. There are various bioavailability and protective effects between these two forms. Aging is a complex biological phenomenon which involves free radicals and oxidative stress. Brain is more susceptible and vulnerable to oxidative damage. Se is involved in the conservation of functional brain activity and protects against the oxidative-stress-related brain disorders. The purpose of this study was to understand the the effects of organic and inorganic selenium compounds proteomic expression and oxidative stress in aged mice brain. The 69-73 weeks old female BALB/c mice were used as animal model. Mice were divided into 3 groups：(1) old control group (OC group; 0.1 mg Se equivalent/kg diet)；(2) inorganic selenium group (selenite, SE group; 0.5 mg Se equivalent/kg diet)；(3) organic selenium group (selenium yeast, SY group; 0.5 mg Se equivalent/kg diet). In addition, the 10 weeks young female BALB/c mice was added as the young control group (YC group; 0.1 mg Se equivalent/kg diet). The mice were fed the test diets for 14 weeks. In mice brains and livers, monoamine oxidase B (MAO-B) enzyme activity in mice brains showed a significant increase in OC group compared with YC group, and showed a significant decrease in the SE and SY groups but no significant change could be noted in the mice livers. However, MAO-B protein expression no significant alteration in the mice brains. The plasma total antioxidant capacity of OC group was significantly decrease than YC group, and SE and SY groups was significantly higher than OC group. In mice brains and livers, the lipid peroxidation and the protein oxidation levels showed a considerable increase in OC group, and showed a significant decrease in the SE and SY groups. Age-related change of the enzymic antioxidants and the non-enzymic antioxidants in brains and livers were also observed. Selenium administration showed significant improvement the enzymic and non-enzymic antioxidant status. In addition, protein expression in brains from organic and inorganic selenium-treated mice was also compared using two dimensional electrophoresis. It was found 18 protein spots expression in brains was differentially regulated between YC and OC group. And aged mice after organic and inorganic selenium administration were found 12 and 18 protein spots, respectively. Particularly, A and C spots protein expression of OC group images was showed significantly increase than YC group, B and D spots protein expression of OC group images was showed significantly decrease than YC group, but A spots protein expression of SE and SY groups images were showed significantly decrease, B spot protein expression of SE group images was showed significantly increase, C spot protein expression of SY group images was showed significantly decrease, D spot protein expression of SY group images was showed significantly increase. The identity of the differentially expressed proteins may help us understand the mechanism of selenium administration. In conclusion, selenium antioxidant properties would be beneficial in ameliorating the age-associated oxidative state. Our results are discussed in context of the role of oxidative stress as one of the important mechanisms of the inorganic selenium and organic selenium supplementation in aged mice brains