臺灣博碩士論文加值系統

樟芝（Antrodia cinnamomea）是台灣特有的藥用真菌，在國內是已許可上市的功能性保健食品。本篇研究是藉由蛋白質體來觀察A. cinnamomea BCRC 35396菌株在液態發酵期間菌絲體生理及代謝上所發生的變化，並進一步的來探討其調控的機制。實驗利用二維電泳建構出不同生長階段之樟芝菌絲體蛋白質圖譜，並比較之間的蛋白質表現。實驗中觀察到，在exponential phase中phosphoribosylaminoimidazole-succinocarboxamide synthase、purine nucleotide biosynthesis-related protein、orotidine 5'-phosphate decarboxylase和elongation factor 2等蛋白皆為正調控表現，推測這個階段的代謝路徑可能著重於核苷酸和蛋白質的生合成。在late-stationary phase則觀察到pyruvate dehydrogenase complex E1-α subunit、squalene synthetase、氧化還原和DNA修復相關蛋白有正調控的表現，由pyruvate dehydrogenase complex E1-α subunit和squalene synthetase正調控的表現推測麥角固醇和三萜類的生合成可能是藉由改變acetyl-CoA的代謝庫來進行調控，先前研究指出，進入穩定生長期後，培養基的養分會逐漸減少並伴隨著大量活性氧的產生，而活性氧具有細胞毒性，會損害DNA、脂質和蛋白質，因此維持氧化還原平衡及DNA修復相關蛋白表現的正調控顯示細胞可能會透過相關調節的機制以維持代謝、氧化還原的平衡與對抗活性氧及氧化壓力。由上述實驗結果可了解樟芝發酵過程中之代謝調節機制，並提供蛋白質體應用於不同培養基組成和發酵條件下，探討微生物代謝生合成機制的可能性。蟬花（Cordyceps sobolifera）為蟲草的一種是稀有且奇特的藥用真菌，而蟲草自古以來就廣泛地運用在疾病的治療上。先前研究指出，蟬花具有保護腎小球功能免受發炎造成之損害、減輕腎間質損傷、促進腎小管上皮細胞修復、恢復腎小管功能，以及促進蛋白質的合成，糾正負氮平衡的作用。本研究以MTT試驗評估蟬花萃取物對腎細胞株之保護作用，實驗中使用具腎毒性之藥物cyclosporin A及蟬花萃取物分別處理及共同處理NRK-52E大鼠腎小管上皮細胞株24小時並觀察細胞之存活率。實驗結果顯示，以濃度30 μM cyclosporin A處理時，細胞存活率約為46.6 %接近半致死濃度，而以濃度250 μg/ml蟬花萃取物與濃度30 μM cyclosporin A共同處理時，細胞存活率提升為69.7 %，證實蟬花萃取物能保護cyclosporin A誘導之NRK-52E腎細胞株的損傷。實驗中並利用二維電泳建構NRK-52E腎細胞株之蛋白質圖譜比較控制組、濃度30 μM cyclosporin A單一處理組，以及濃度250 μg/ml蟬花萃取物與濃度30μM cyclosporin A共同處理組之間蛋白質表現的差異，且進一步探討蛋白質表現變化之意義及其功能性。在cyclosporin A單一處理組鑑定到一些對於細胞生理較為重要的蛋白，像是細胞骨架、細胞週期、細胞增生與分化相關蛋白有負調控的表現，而在蟬花萃取物和cyclosporin A共同處理組則發現這些蛋白的表現量有回復的情形。由上述實驗結果有助於了解蟬花萃取物保護cyclosporin A誘導之NRK-52E腎細胞株損傷的作用機制。

Antrodia cinnamomea is a unique medicinal fungus that has been used as a functional food in Taiwan. The concepts of this study are investigating the physiological and metabolic changes through observing the proteome of A. cinnamomea BCRC 35396 while it is fermenting. In order to compare the expression of proteins among each growth phase, we constructed 2-DE patterns of intercellular proteins from each growth phase of A. cinnamomea cells during fermentation. We observed some proteins such as phosphoribosylaminoimidazole-succinocarboxamide synthase, purine nucleotide biosynthesis-related protein, orotidine 5'-phosphate decarboxylase and elongation factor 2 showed up-regulation. Therefore, we inferred that the main metabolic pathway during exponential phase may emphasize the biosynthesis of nucleotides and proteins. The late-stationary phase signature revealed that up-regulation of pyruvate dehydrogenase complex E1-α subunit, squalene synthetase, redox-related and DNA repair-related proteins. The up-regulation of pyruvate dehydrogenase complex E1-α subunit and squalene synthetase may indicate that the biosynthesis of ergosterol and triterpenoids were mediated by switching the metabolic pool of acetyl-CoA, and the previous study indicated that in the late-stationary phase, A. cinnamomea cells have to face the reducing of medium nutritious and the increasing of the reactive oxygen species. The up-regulation of redox-related and DNA repair-related enzymes in the A. cinnamomea cells may reflect to the cells attempted to maintain metabolic redox homeostasis through regulating and coping with reactive oxygen species and nutritional stress. The foregoing results could understand the mechanism of metabolic regulation, and provide the applicability of proteome. Cordyceps sobolifera belongs to Cordyceps and is a rare and exotic medicinal fungus. It has been demonstrated that C. sobolifera has effects on protecting glomerular function from inflammation, decreasing the damage in renal interstitial cells and promoting the repair of renal tubule epithelial cells. This study was divided into two parts. The first part was using MTT assay to examine the effects of C. sobolifera extract (CSE), cyclosporin A (CSA) treated respectively and both two cotreated in NRK-52E rat kidney proximal renal tubule cell line for 24hrs. The data showed that 30 μM CSA -treated for 24hrs, the viability of NRK-52E cells were 46.6 % close to LC50, and when 250 μg/ml CSE and 30 μM CSA-cotreated, the viability of NRK-52E cells rose to 69.7 %. Results indicated that CSE could protect NRK-52E cells from the CSA-induced damage. The second part was to construct 2-DE patterns of intercellular proteins from NRK-52E cells for observing the changes of proteins and discussing the significance of these changes during the different treatments. We observed that some main proteins presented down-regulation in NRK-52E cells which were related to cell cycle, cell proliferation and differentiation while 30 μM CSA -treated for 24hrs, and the expression of these proteins increased in NRK-52E cells while 250 μg/ml CSE and 30 μM CSA-cotreated for 24hrs. The foregoing results may be helpful to understand the mechanism of CSE could protect NRK-52E cells from the CSA-induced damage.

目錄……………………………………………………………………Ⅰ
圖目次…………………………………………………………………Ⅵ
表目次…………………………………………………………………Ⅶ
中文摘要………………………………………………………………Ⅷ
英文摘要………………………………………………………………Ⅹ
前言……………………………………………………………………1
文獻回顧………………………………………………………………3
壹、樟芝………………………………………………………………3
一、樟芝命名…………………………………………………………3
二、分類地位…………………………………………………………4
三、簡介………………………………………………………………4
四、功效成份…………………………………………………………5
（一）多醣體…………………………………………………………6
（二）三萜類…………………………………………………………6
（三）腺苷……………………………………………………………6
（四）超氧歧化酵素…………………………………………………7
（五）麥角固醇………………………………………………………7
貳、蟬花………………………………………………………………8
一、分類地位…………………………………………………………8
二、大小蟬花之區分…………………………………………………8
三、典籍記載…………………………………………………………8
四、生長型態及生態特性……………………………………………9
五、研究現況…………………………………………………………9
參、蛋白質體學………………………………………………………10
一、簡介………………………………………………………………10
二、研究範疇…………………………………………………………10
（一）表現性蛋白質體學……………………………………………10
（二）功能性蛋白質體學……………………………………………11
（三）生產性蛋白質體學……………………………………………11
（四）結構性蛋白質體學……………………………………………12
（五）細胞位圖蛋白質體學…………………………………………12
（六）生物資訊蛋白質體學…………………………………………12
三、操作技術…………………………………………………………12
（一）蛋白質的分離…………………………………………………12
（二）蛋白質的鑑定…………………………………………………13
（三）資料庫的搜尋比對……………………………………………13
材料方法………………………………………………………………14
一、實驗材料…………………………………………………………14
（一）實驗樣品來源…………………………………………………14
（二）實驗藥品………………………………………………………14
（三）實驗器材及儀器設備…………………………………………16
二、實驗方法…………………………………………………………17
（一）樟芝實驗………………………………………………………17
1.樟芝菌種活化與液態發酵培養……………………………………17
2.pH值測定……………………………………………………………18
3.菌絲體產量變化測定………………………………………………18
4.發酵液殘糖量測定…………………………………………………18
5.發酵產物總多醣含量測定…………………………………………18
6.腺苷之萃取與分析…………………………………………………19
7.麥角固醇之萃取與分析……………………………………………19
8.粗三萜類之萃取與分析……………………………………………19
9.樟芝菌絲體蛋白質樣品製備………………………………………20
9.1.蛋白質樣品製備前處理…………………………………………20
9.2.TCA及acetone 蛋白質沈澱處理………………………………20
9.3.蛋白質萃取………………………………………………………20
9.4.蛋白質萃取液之定量……………………………………………21
（二）細胞實驗………………………………………………………21
1.細胞株培養條件……………………………………………………21
2.繼代培養……………………………………………………………22
3.MTT試驗分析………………………………………………………22
4.蛋白質萃取…………………………………………………………23
5.細胞蛋白質萃取液之定量…………………………………………24
（三）二維膠體電泳分析蛋白質實驗………………………………24
1.一維電泳樣品前處理………………………………………………24
2.一維等電焦集電泳…………………………………………………24
3.SDS-PAGE電泳膠片製作…………………………………………25
4.二維聚丙烯醯胺膠體電泳…………………………………………25
5.蛋白質之固定及染色………………………………………………26
（四）蛋白質身份鑑定………………………………………………27
1.膠片掃描……………………………………………………………27
2.軟體比對……………………………………………………………27
3.蛋白質膠內水解……………………………………………………27
4.質譜分析與資料庫搜尋……………………………………………28
結果……………………………………………………………………29
壹、以蛋白質體探討樟芝菌絲體液態發酵過程……………………29
一、菌絲體產量、發酵液殘糖量及pH值之表現……………………29
二、功效成份含量之變化……………………………………………29
三、蛋白質表現及身分鑑定…………………………………………30
貳、以蛋白質體探討蟬花萃取物保護cyclosporin A誘導之
NRK-52E腎細胞的損傷………………………………………………31
一、MTT試驗分析……………………………………………………31
二、蛋白質表現與身分鑑定…………………………………………32
討論……………………………………………………………………33
壹、以蛋白質體探討樟芝菌絲體液態發酵過程……………………33
貳、以蛋白質體探討蟬花萃取物保護cyclosporin A誘導之
NRK-52E腎細胞株的損傷……………………………………………37
參考文獻………………………………………………………………62

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