臺灣博碩士論文加值系統

已知蜜蜂(Apis mellifera)可當作研究老化的模式動物，蜂王的壽命約是工蜂的12倍。本研究以老化指標研究兩者老化過程中的差異。我們使用顯微鏡、生化分析及分生技術研究工蜂與蜂王腹部滋養細胞及脂肪細胞中老化指標之變化。
結果顯示，工蜂與蜂王老化指標相似處為β-半乳糖苷酶、脂質過氧化、蛋白質氧化、過氧化氫酶以及麩光甘肽過氧化酶的表現量隨年紀增加而增加；而SirT1的表現量則相反。端粒酶活性及端粒長度無顯著差異。
工蜂與蜂王老化指標相異處為，在工蜂之超氧陰離子及過氧化氫、超氧歧化酶(MnSOD、CuZnSOD)活性、Catalase表現量隨年齡增加而下降；而FOXO、JNK與MnSOD表現量隨年齡增加而增加。蜂王之脂褐質、JNK、Catalase表現量隨年齡上升無顯著差異。
由上述結果發現在工蜂與蜂王之老化指標表現量在代謝廢物堆積、氧化壓力、抗氧化酵素效率、與代謝相關基因表現相異，這些相異之處，可提供未來進一步研究蜂王長壽之原因，進而提供未來研究延緩老化的基礎。

Honeybees (Apis mellifera) have served as an animal model for the study of aging. The lifespan of queen bees is 12-fold longer than that of worker bees.This study differentiates the differences of age-related molecules between worker bees and queen bees by using histochemical, biochemical, and genetic techniques.
The results showed that senescence-associated -galactosidase, lipid peroxidation, protein oxidation, glutathione peroxidase activity and catalase activity were increased with age, whereas SirT1 expression is decreased with age in worker bees and queen bees. However, telomerase activity and telomere length have no significant difference.
The results also showed that the expression of some age-related molecules was different between worker bees and queen bees. Reactive oxygen species(O2‧- and H2O2), superoxide dismutase activity(MnSOD and Cu/ZnSOD) ,Catalase expression are decreased with age, whereas the expression of FoxO, MnSOD and JNK was increased with age in worker bees. Besides the expression of Lipofuscin granules, JNK and Catalase expression has no significant difference with worker bees, the expression of remainder is reversely proportioned to worker bees in queen bees.
Collectively, some age-related molecules expressed similar tendencies; others did different tendencies between worker bees and queen bees. The different expression (Waste accumulation,ROS level,Antioxidant enzyme efficiency,Gene regulation)of age-related molecules between worker bees and queen bees laid a foundation to further study the mechanism of aging and anti-aging.

目錄
指導教授推薦書 ……………………………………………………i
論文口試委員審定書………………………………………………ii
長庚大學授權書 …………………………………………………iii
誌謝 …………………………………………………………………iv
中文摘要………………………………………………………………v
英文摘要………………………………………………………………vi
目錄 …………………………………………………………………vii
圖表目錄 ……………………………………………………………xiv
第一章 序論 …………………………………………………1
1.1老化研究背景………………………………………………………1
1.2 老化指標物質………………………………………………………2
1.2.1 Lipofuscin granules(脂褐質) …………………………2
1.2.2 Senescence associated β-galactosidase
(SA-β-Gal，SA-β-半乳糖苷酶) ……………………………4
1.3 氧化壓力與抗氧化酵素……………………………………………5
1.3.1 活性氧(reactive oxygen species，ROS)………………5
1.3.2 lipid peroxidation(脂質過氧化)………………………6
1.3.3 protein oxidation(蛋白質氧化) ………………………7
1.3.4超氧歧化酶(Superoxide Dismutase,SOD) ………………8
1.3.5 過氧化氫酶(Catalase)……………………………………9
1.3.6 麩光甘肽過氧化酶(Glutathione peroxidease，GPx)…11
1.4端粒與端粒酶………………………………………………………11
1.4.1 Telomere length(端粒長度) ……………………………11
1.4.2 Telomerase activity(端粒酶活性) ……………………13
1.5參與代謝之訊號傳遞………………………………………………14
1.5.1 SirT1(Sirtuin1) signal…………………………………14
1.5.2 FOXO (Forkhead box o) signal…………………………16
1.5.3 JNK (c-Jun N-termial kinase) …………………………18
1.6模式動物(Animal model)
1.6.1世界各大蜜蜂飼養品系……………………………………19
1.6.2使用義大利蜂為模式動物…………………………………21
1.6.3蜂王…………………………………………………………22
1.6.4蜜蜂腹部細胞………………………………………………26
第二章 論文研究目的 ……………………………………………27
第三章 實驗材料與方法 …………………………………………28
3.1樣品取得……………………………………………………………29
3.2 萃取蛋白質 ………………………………………………………29
3.3 Lipofuscin granules……………………………………………29
3.4 SA-β-gal…………………………………………………………30
3.5 ROS 測定 …………………………………………………………30
3.5.1超氧化物自由基(Superoxide，O2−.)測定 ………………30
3.5.2過氧化氫(Hydrogen peroxide，H2O2)測定………………31
3.6 lipid peroxidation assay………………………………………33
3.7 protein oxidation assay ………………………………………33
3.8 SOD activity assay………………………………………………34
3.9 Catalase activeity assay………………………………………35
3.10 GPx activeity assay……………………………………………35
3.11 Western blotting(西方墨點法) ………………………………36
3.12 TRAP assay (Telomere Repeat Amplification Protocol) …37
3.13 萃取genomic DNA ………………………………………………38
3.14 Southern blotting(南方墨點法) ……………………………39
3.15 統計方法…………………………………………………………39

第四章 實驗結果……………………………………………………40
4.1工蜂部份……………………………………………………………40
4.1.1 脂褐質(lipofuscin granules)分析結果 ……………41
4.1.2β-半乳糖苷酶(SA-β Gal)分析結果 …………………41
4.1.3 超氧陰離子(O2‧-)分析結果 ……………………………41
4.1.4過氧化氫(H2O2)分析結果…………………………………41
4.1.5 脂質過氧化程度(lipid peroxidation) 分析結果 …41
4.1.6 蛋白質氧化程度(protein oxidation) 分析結果……42
4.1.7 過氧化氫酶(Catalase)活性分析結果…………………42
4.1.8麩光甘肽過氧化酶(Glutathione peroxidease，GPx)活性分析結果……………………………………………………42
4.1.9 銅-鋅-超氧歧化酶(copper-zinc superoxide dismutase,
Cu-ZnSOD)活性分析結果…………………………………43
4.1.10 錳-超氧歧化酶(Manganese superoxide dismutase,MnSOD) 活性分析結果………………………43
4.1.11 端粒酶活性(Telomerase activity)分析結果………43
4.1.12 端粒長度(Telomere length) 分析結果 ……………44
4.1.13 Forkhead box-O(FOXO)表現量分析結果………………44
4.1.14 SirT1(Sirtiun 1)表現量分析結果……………………44
4.1.15 JNK(c-Jun N-terminal kinase)表現量分析結果……45
4.1.16 MnSOD表現量分析結果…………………………………46
4.1.17 Catalase表現量分析結果 ……………………………46
4.2蜂王部份……………………………………………………………47
4.2.1脂褐質(lipofuscin granules)分析結果………………47
4.2.2β-半乳糖苷酶(SA-β Gal)分析結果 …………………47
4.2.3 超氧陰離子(O2‧-)分析結果 ……………………………47
4.2.4過氧化氫(H2O2)分析結果…………………………………48
4.2.5 脂質過氧化程度(lipid peroxidation)分析結果……48
4.2.6 蛋白質氧化程度(protein oxidation)分析結果 ……48
4.2.7過氧化氫酶(Catalase)活性分析結果 …………………49
4.2.8麩光甘肽過氧化酶(Glutathione peroxidease，GPx)活性分析結果…………………………………………………49
4.2.9錳-超氧歧化酶(Manganese superoxide dismutase,MnSOD)活性分析結果………………………………………………50
4.2.10 銅-鋅-超氧歧化酶(copper-zinc superoxide dismutase,
Cu-ZnSOD)活性分析結果 …………………………………50
4.2.11 端粒酶活性(Telomerase activity)分析結果………51
4.2.12端粒長度(Telomere length)分析結果 ………………51
4.2.13 Forkhead box-O(FOXO)表現量分析結果………………51
4.2.14 SirT1(Sirtiun 1)表現量分析結果……………………52
4.2.15 JNK(c-Jun N-terminal kinase)表現量分析結果……52
4.2.16 MnSOD表現量分析結果…………………………………52
4.2.17 Catalase表現量分析結果 ……………………………53
4.3工蜂與蜂王老化指標差異…………………………………………54
4.3.1 脂褐質(lipofuscin granules)差異 …………………54
4.3.2 β-半乳糖苷酶(SA-β Gal)活性差異…………………54
4.3.3 超氧陰離子(O2‧-)差異 …………………………………54
4.3.4 過氧化氫(H2O2)差異 ……………………………………54
4.3.5 脂質過氧化程度(lipid peroxidation)以及蛋白質氧化程度(protein oxidation)差異 …………………………55
4.3.6 過氧化氫酶(Catalase)與麩光苷肽過氧化酶(Glutathione peroxidease，GPx)活性差異……………56
4.3.7 超氧歧化酶(superoxide dismutase)活性差異………57
4.3.8 端粒酶活性(Telomerase activity)與端粒長度(Telomere length)差異 …………………………………57
4.3.9 Forkhead box-O(FOXO)表現量差異……………………57
4.3.10 SirT1(Sirtiun 1)表現量差異…………………………57
4.3.11 JNK(c-Jun N-terminal kinase)表現量差異…………58
4.3.12 MnSOD表現量分析結果…………………………………58
4.3.13 Catalase表現量分析結果 ……………………………58
第五章 討論 …………………………………………………60
5.1工蜂與蜂王細胞內老化相關指標物質之變化……………………60
5.2工蜂與蜂王細胞內氧化壓力與抗氧化酵素之變化………………63
5.3 工蜂與蜂王端粒酶活性與端粒長度之變化 ……………………65
5.4工蜂與蜂王參與代謝之基因表現變化差異………………………67
5.4.1工蜂細胞內之代謝之基因表現變化………………………67
5.4.2蜂王細胞內參與代謝之基因變化…………………………68
5.5工蜂與蜂王細胞內抗氧化酵素效率差異…………………………70
第六章 結論 …………………………………………………72
圖表……………………………………………………………74
參考文獻 ……………………………………………………101
附錄
附錄一、工蜂與蜂王老化指標比較圖
附錄二、光學顯微鏡下不同年齡之工蜂細胞型態
附錄三、光學顯微鏡下不同年齡之蜂王細胞型態
附錄四、10~40日齡之工蜂外觀變化
附錄五、使用SPSS軟體進行多重比較方法
附錄六、本論文中圖表製作方式
圖表目錄
圖一、不同階級(caste)之蜜蜂外觀差異 ……………………………74
圖二、不同年齡工蜂之外觀差異………………………………………75
圖三、不同年齡蜂王之外觀差異………………………………………75
圖四、蜜蜂腹部細胞分布情形…………………………………………76
圖五、不同年齡工蜂之脂褐質(lipofuscin granules)分析結果…77
圖六、不同年齡工蜂之SA-β-半乳糖苷酶(SA-β-gal)分析結果…78
圖七、不同年齡工蜂之超氧自由基(O2‧-)分析結果…………………79
圖八、不同年齡工蜂之過氧化氫(H2O2)分析結果……………………80
圖九、不同年齡工蜂之脂質過氧化(lipid peroxidation)分析結果 ………………………………………………………………81
圖十、不同年齡工蜂之蛋白質氧化(protein oxidation)分析結果 ………………………………………………………………82
圖十一、不同年齡工蜂之過氧化氫酶(catalase)、麩胱甘肽過氧化酶(GPx)、超養歧化酶(SOD)分析結果 …………………………83
圖十二、不同年齡工蜂之端粒酶活性(Telomerase activity)、端粒長度(Telomere length)分析結果 …………………………84
圖十三、不同年紀工蜂之端粒長度計算分析結果……………………85
圖十四、不同年齡工蜂之FOXO表現量分析結果(70kDa)……………85
圖十五、不同年齡工蜂之SirT1表現量分析結果(82kDa) …………86
圖十六、不同年齡工蜂之JNK表現量分析結果(54/46kDa)…………86
圖十七、不同年齡工蜂之MnSOD表現量分析結果(24kDa)……………87
圖十八、不同年齡工蜂之Catalase表現量分析結果(64kDa)………87
圖十九、不同年齡蜂王之脂褐質(lipofuscin granules)分析結果 ……………………………………………………………88
圖二十、不同年齡蜂王之SA-β-半乳糖苷酶(SA-β-gal)分析結
果 ……………………………………………………………89
圖二十一、不同年齡蜂王之超氧自由基(O2‧-)分析結果……………90
圖二十二、不同年齡蜂王之過氧化氫(H2O2)分析結果………………91
圖二十三、不同年齡蜂王之脂質過氧化(lipid peroxidation)分析 …………………………………………………………92
圖二十四、不同年齡蜂王之蛋白質氧化(protein oxidation)分析
結果 ………………………………………………………93
圖二十五、不同年齡蜂王之過氧化氫酶(catalase)、麩胱甘肽過氧化酶(GPx)、超養歧化酶(SOD)分析結果……………………94
圖二十六、不同年齡蜂王之端粒酶活性(Telomerase activity)、端粒長度(Telomere length)分析結果……………………95
圖二十七、不同年紀蜂王之端粒長度計算分析結果 ………………96
圖二十八、不同年齡蜂王之FOXO表現量分析結果(70kDa) …………96
圖二十九、不同年齡蜂王之SirT1表現量分析結果(82kDa)…………97
圖三十、不同年齡蜂王之JNK表現量分析結果(54/46kDa) …………97
圖三十一、不同年齡蜂王之MnSOD表現量分析結果(24kDa)…………98
圖三十二、不同年齡蜂王之Catalase表現量分析結果(64kDa) ……98
表一、不同年齡工蜂老化指標變化……………………………………99
表二、不同年齡蜂王之老化指標變化 ………………………………100

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