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膜衣包覆技術生產參數之建立
No full text保健素材有別於一般食品，其中的活性成分脆弱，易受到外在因素破壞而失活，此外，保健素材大多帶有不悅的味道，不容易被人接受當作一般休閒食品食用。因此，想將保健素材應用於一般食品當中，必須先進行適當的加工，一方面保護保健活性成分，另一方面隔絕不悅味道。
本研究室過去有關膜衣包覆技術及味道修飾的研究，能夠對保健素材達到保護和味道修飾的效果，製作材料與流程適用於食品法規，具有應用在食品工業上的潛力。本研究以這項技術當作基礎，透過電子顯微鏡、包覆效果測定及控制釋放實驗，收集此技術相關的實驗數據和操作經驗，嘗試為這項膜衣包覆技術建立較佳的生產模式與流程。
本研究針對樣品前處理、包覆過程及後處理等步驟，透過實驗得到各個步驟較佳的操作參數。最後，本研究以檸檬酸作為指標，檢驗經過包覆的成品在2分鐘內(20、40、60和120秒)的釋放效果。包覆後的粉末可以明顯(p < 0.05)降低核心物質在至少兩分鐘內所釋放的物質濃度，此外，未包覆的粉末在20秒時的釋放濃度，略高於包覆後的粉末在120秒時的釋放濃度，能夠推估包覆技術成品可以讓核心物質的釋放至少延緩100秒以上。Functional foods have become popular and have made the food industry lots of profit in recent years. This has made functional food the focus of development in food industry. Companies are searching for new functional materials to make new products. The bioactivities of many functional materials are easily affected by different environmental factors. Techniques that can barrier the active agent in functional materials from outside factors are being applied to solve this problem. Encapsulation is one of the main techniques employed to solve these problems.
Our laboratory has previously developed different food particle coating techniques and has found their potential application in food processing. The technique can form a food-based barrier to protect an active agent from outside factors to attain taste masking, controlled release, and moisture proofing. A practical approach and production procedure was established in this study.
Several steps include pre-treatment, coating process, and post-treatment were studied to determine appropriate operating parameters of the coating technique. At last, citric acid was used as a marker to evaluate the releasing ability of the coated particles at 4 different time points such as 20, 40, 60, and 120 seconds.
At the conclusion of the study, some appropriate operating parameters for this film coating technique have been determined. Finally, it was found that the coated particles could delay the dissolution more effectively (p < 0.05) at every time point as the amount of citric acid being released from the coated particles at 120 seconds was comparable to that released from the uncoated particles at 20 seconds.中文摘要 i
英文摘要 ii
目錄 iv
表次 vi
圖次 vii
1. 前言 1
1.1 膠囊化技術 1
1.2 膠囊化技術在食品上之應用 1
1.2.1 不悅味道的遮蔽 4
1.2.2 改善活性物質穩定性 5
1.2.3 控制釋放 6
1.3 可應用於食品之膠囊化方法 6
1.4 應用於食品產業之膠囊材料 9
1.5 檢測膠囊化技術之方法 12
2. 研究目的 14
3. 材料與方法 15
3.1 研究實驗架構 15
3.2 核心粉末 15
3.3 包覆材料 15
3.4 薄膜包覆 15
3.4.1 前處理 18
3.4.2 包覆材料與核心粉末比例 18
3.4.3 後處理 18
3.4.4 重複 18
3.5 控制釋放實驗 18
3.6 掃描式電子顯微鏡 19
3.7 粒徑分析 19
3.8 統計方法 19
4. 結果與討論 20
4.1 核心粉末及包覆材料之製備 20
4.2 理論包覆量 20
4.3 薄膜包覆 21
4.3.1 核心粉末大小之選用 23
4.3.2 前處理階段之水合效果變化 27
4.3.3 包覆材料混合結果 31
4.4 控制釋放結果 34
4.4.1 未包覆檸檬酸粉末的釋放結果 34
4.4.2 包覆成品的釋放結果 34
4.4.3 包覆成品的保護效果 36
4.5 較佳的膜衣包覆流程 42
5. 總結論 46
6. 參考文獻 4
文心蘭中 Oncidium SUPPRESSOR OF CONSTANS OVEREXPRESSION 1(OnSOC1)基因之特性與功能性分析
No full text從營養期過渡至開花階段，是植物繁殖的重要關鍵。植物中調控開花周期 主要有四個途徑:光週期、春化作用、植物自主性以及吉貝素。在阿拉伯芥中， MADS-box 蛋白質 SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1)扮演著 整合多種開花信號進而調控營養期到生殖發育過程的角色。在本研究中，我們從 南茜品種文心蘭(Oncidium Gower Ramsey)選殖出 SOC1 的同源基因(OnSOC1)， 對 其 進 行 其 功 能 性 分 析 。 研 究 結 果 顯 示 ， 大 量 表 現 OnSOC1 和 強 烈 活 化 OnSOC1-VP16 的轉基因植物中呈現提早開花的現象，而在大量表現強烈抑制 OnSOC1-SRDX 則呈現延遲開花的現象。經由基因表現量分析，發現在大量表現 OnSOC1 之提早開花植株中，開花調控相關基因 AGL24 及 LFY 表現量都有所提 高，表示 OnSOC1 藉由調控 AGL24 及 LFY 造成提早開花，在開花時間的調控上 與 SOC1 有類似的功能。此外與 SOC1 蛋白質相同，OnSOC1 亦可以與 AGL24 形成蛋白質異質二聚體。除了影響開花時間外，OnSOC1-SRDX 也會造成花器延 緩老化及凋落的現象。本研究顯示在演化上，SOC1 同源基因在促進植物開花的 功能上是同時保留在蘭科植物上。因此 OnSOC1 未來可被用來調控文心蘭開花 時間之實際應用上。Proper timing of the transition from vegetative stage to flowering is critical to the reproductive success of plants. The floral transition in Arabidopsis is regulated by four flowering pathways: the photoperiod, vernalization, autonomous, and gibberellin dependent pathway. SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) encodes a MADS-box protein that plays an essential role in integrating multiple flowering signals to regulate the transition from vegetative to reproductive development in the model plant Arabidopsis. To investigate the function of its homologue in Orchid, we cloned OnSOC1 from Oncidium Gower Ramsey. In this study, further functional analysis of the OnSOC1 in transgenic Arabidopsis was performed. The flowering time was promoted in 35S::OnSOC1 and 35S::OnSOC1-VP16 (fused an activation domain) transgenic plants whereas 35S::OnSOC1-SRDX (fused a repressing domain) caused the delay of flowering. These results indicated that OnSOC1 acted as an activator in promoting flowering in transgenic Arabidopsis. The promotion of flowering time in 35S::OnSOC1 Arabidopsis was due to the up-regulation of AGL24 and LEAFY expression. Furthermore, similar to SOC1, OnSOC1 could form heterodimers with Arabidopsis AGL24. 35S::OnSOC1-SRDX also caused significant delay of flower senescence and abscission in transgenic Arabidopsis. Our results revealed that SOC1-like genes play an evolutionarily conserved role in promoting flowering in the Orchidaceae family. Thus, OnSOC1 isolated from Oncidium Gower Ramsey could be used as an important target for genetic manipulation of flowering time in Orchid.摘要……Ⅰ
Abstract……Ⅱ
前言……1
材料與方法……5
結果……14
1從文心蘭中選殖阿拉伯芥開花調控同源基因OnSOC1……14
1.1文心蘭中開花基因OnSOC1之選殖……14
1.2文心蘭中開花基因OnSOC1序列及演化樹之分析……15
1.3文心蘭中開花基因OnSOC1之各部位表現……15
2分析OnSOC1對阿拉伯芥中之開花時間的影響……16
2.1OnSOC1、OnSOC1-SRDXR及OnSOC1-VP16的分子選殖與載體構築……17
2.2異位表現OnSOC1、OnSOC1-SRDX及OnSOC1-VP16在阿拉伯芥中造成開花時間之差異……17
2.3大量表現OnSOC1與OnSOC1-VP16之轉植株會促進AGL24LFY表現、量上升進而造成提早開花……18
2.4OnSOC1-SRDX轉植株內之離層及老化相關基因表現量下降導致延緩花器的老化與脫落……18
3比較文心蘭與阿拉伯芥間MADS-box蛋白SOC1、SVP與AGL24交互作用的差異……19
3.1酵母菌之載體構築、轉型及雜交作用……20
3.2OnSOC1與AtAGL24之間的相互作用並不顯著……20
3.3OnSOC1與文心蘭之開花調控基因OnSVP1/2的相互作用……21
4探討SOC1對阿拉伯芥的老化之影響……21
4.1AtSOC1、AtSOC1-SRDXR及AtSOC1-VP16的分子選殖與載體構築……22
4.2異位表現AtSOC1-SRDXR轉植株內之離層及老化相關基因表現量下降導致延緩花器的老化與脫離……22
4.3大量表現AtSOC1及AtSOC1-VP16在阿拉伯芥造成老化相關基因得表現量上升導致花器的提早老化……23
5.探討OnSOC1對於文心蘭中開花時間的影響……23
5.1OnSOC1RNAi之載體構築……23
討論……25
參考文獻……29
圖表……3
人類6-磷酸葡萄糖異構酶與鳥苷三磷酸結合位之探討
No full text6-磷酸葡萄糖異構酶 (Phosphoglucose isomerase, PGI) 是一個多功能的蛋白質，主要作用為催化 6-磷酸葡萄糖 (glucose-6-phosphate, G6P) 與 6-磷酸果糖 (fructose-6-phosphate, F6P) 間的異構化作用。PGI 廣泛存在於各物種內，參與在糖解代謝、糖質新生等重要生理作用中。鳥苷三磷酸 (Guanosine triphosphate, GTP) 為蛋白質合成及糖質新生作用中的主要能量來源，也是代謝作用中基質的激活因子。從先前研究中發現，GTP 會與人類 PGI (human PGI, hPGI) 結合，但並不會與大腸桿菌 (Escherichia coli PGI, ePGI) 結合。本實驗研究目的為探討 hPGI 與 GTP 之胺基酸結合位置，並進一步了解其結合後之特性。首先在 hPGI 含有 MgCl2的條件中部分 GTP 會被水解產生 GDP。利用數種磷酸糖類干擾 [α-32P]-GTP 與 hPGI 的交聯作用。由結果發現 GTP、F6P 及 G6P 等實驗組會影響 [α-32P]-GTP 與 hPGI 的結合。從酵素動力學得知，GTP 與 6-phosphate-gluconate (6P-GA) 對 hPGI 的作用為競爭性抑制，經換算之 Ki 值分別為 63 μM 及 12 μM。Yonetani-Theorell 雙抑制實驗結果發現，GTP 與 6P-GA 間的 α值約為1.7，介於∞與1之間，表示GTP 及 6P-GA 會干擾與 hPGI 的結合。由於先前實驗懷疑 hPGI 第 92 個酪胺酸 (Tyrosine, Y) 及第 95 個甘胺酸 (Glycine, G) 殘基可能與 GTP 結合有關，因此利用點突變方式得到 Y92R、G95N 及 Y92R/G95N；另外利用點突變得到 ePGI 的突變蛋白質 R90Y、N93G 及 R90Y/N93G 以進行與 GTP 結合實驗。結果發現，hPGI Y92R/G95N 對 GTP 的結合力減弱。利用分子模擬 (Molecular modelling) 預測 GTP 結合特定之 hPGI 位置的結構模型發現，其獲得最低自由能為 -10.7 kcal/mol；另外 R96 及 Q512 皆同時出現在三個預測結構中。蛋白質結晶實驗中，三種結晶條件有獲得 hPGI 的蛋白質晶體，但沒有發現 GTP 與 hPGI 結合。X ray 結果發現，有類似磷酸根的結構鑲嵌在 S159、S209、K210、T214、H288 間。Phosphoglucose isomerase (PGI) is a protein with multiple functions. It catalyzes the interconversion between glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). Widely existing in diverse species, PGI acts as an isomerization enzyme in glycolysis and gluconeogenesis pathways. Guanosine triphosphate (GTP) is used as a source of energy for protein synthesis and gluconeogenesis. GTP also played a role as an activator of substrates in metabolic reactions. In a precious study, human PGI was found to interact with GTP, but not with Escherichia coli (E. coil) PGI. The purpose of this study is to investigate which amino acids on the human PGI will interact with GTP and to find out the consequence of the interaction. First of all, hPGI could hydrolyzed GTP when the MgCl2 include in the buffer. Several phosphosugar were used to interfere with the UV-induced [α-32P]-GTP crosslink to human PGI. The results showed that GTP, F6P and G6P could affect the crosslink between [α-32P]-GTP and the protein. Enzyme kinetic studies indicated that both of GTP and 6-phosphate gluconate (6P-GA) are competitive inhibitors with the values of Ki 63 μM and 12 μM, respectively. Yonetani-Theorell double-inhibition experiment found that both GTP and 6P-GA have values of α approximately 1.7 ；suggesting that GTP and 6P-GA could interfere with each other for the binding to human PGI. The previous study also suspected that the 92 tyrosine (Tyrosine, Y) and 95 glycine (Glycine, G) residues may involve the GTP binding. Therefore, mutations of Y92R, G95N and Y92R/G95N were created; on the other hands, mutations of R90Y, N93G and R90Y/N93G in E. coli PGI were created. GTP-binding assay indicating that Y92R/G95N weakens the binding of GTP to human PGI. The GTP binding specified area of hPGI investigating by molecular modelling prediction estimated the minimum Gibbs free energy about -10.7 kcal/mol. R96 and Q512 are simultaneously in the three predicted structure. In the protein crystallization experiments with three crystallization conditions, hPGI protein crystals were collected, but the GTP combination with human PGI was not found. X-ray results showed a similar phosphate structures embedded in the S159, S209, K210, T214 and H288.摘要………………………………………………………………………………………Ⅰ
Abstract ………………………………………………………………………………….Ⅱ
目錄……………………………………………………………………………………....Ⅲ
表目錄…………………………………………………………………………………....Ⅴ
圖目錄…………………………………………………………………………………....Ⅵ
第一章、 前言………………………………………………………………….……......1
第一節、 6-磷酸葡萄糖異構酶之特性…………………………..…….…….......1
第二節、 人類 6-磷酸葡萄糖異構酶之相關研究………………..……................2
一、 促神經細胞生長因子/神經白介素…………………….........................2
二、 自泌性促腫瘤移動因子…………………………………..…….……...2
三、 細胞促分化成熟介子……………………………………..……............2
四、 遺傳性非球形溶血性貧血……………………………..……………....3
第三節、 鳥苷三磷酸之特性……………………………………………..............3
第四節、 相關酵素與三磷酸核苷作用之研究………………………..................4
一、 PFK及 G6PDH與三磷酸核苷之研究…………...…...........................4
二、 AGE 與 ATP 之結合作用研究………………………………………...4
三、 人類 6-磷酸葡萄糖異構酶與鳥苷三磷酸結合之研究......................... 5
四、 人類 6-磷酸葡萄糖異構酶與鳥苷三磷酸之細胞研究………..……....5
第五節、 6-磷酸葡萄糖異構酶作用之結構特性…………………………..….....6
第六節、 研究動機與目的……………………………...…………………….......7
第二章、 材料與方法…………………………………………………………...…….....8
第一節、 菌株及載體……………………………………………………………......8
第二節、 培養基成分……………………………………………………………......9
第三節、 DNA 重組………………………………………………………………....10
第四節、 點突變基因構築……………………………………………………........13
第五節、 蛋白質製備…………………………………………………………........15
第六節、 GTP 與 PGI 作用特性分析……………………………………………...16
第七節、 酵素活性測試…………………………………………………………....17
第八節、 序列比對…………………………………………………………...….....19
第九節、 蛋白質結構預測………………………………………………………....19
第十節、 蛋白質結晶……………………………………………………………....20
第十一節、蛋白質晶體繞射數據收集及結構模型之建立與修正………………...21
第三章、 結果……………………………………………………………...…………...22
第四章、 討論……………………………………………………………...…………...28
第五章、 參考文獻…………………………………………………………...………...53
第六章、 附件…………………………………………………………………...……...62
表目錄
表 3-1 蛋白質結晶嘗試條件。…………………………………………………...…...32
表 3-2 獲得蛋白質結晶之條件。……....…………………………………………......33
圖目錄
圖 1-1 6-磷酸葡萄糖異構酶催化作用反應。………………………….…………….34
圖 2-1 牛血清蛋白 (Bovine Serum Albumin, BSA) 濃度及吸光 OD595 作標準曲線。….……………………………………………………………………….…………...35
圖 3-1 初萃液及 Ni+-NTA 純化後之人類 6-磷酸葡萄糖異構酶。………………....36
圖 3-2 SP 及 Gel filtration 純化後之人類 6-磷酸葡萄糖異構酶。……....………….37
圖 3-3 利用 TLC 分析 6-磷酸葡萄糖異構酶水解 GTP 能力測試。….…………….38
圖 3-4 利用 NTP、GMP、cGMP、F6P 及 G6P 與 [α-32P] GTP 競爭 hPGI 能力測試。….……………………………………….……………………………….………......39
圖 3-5 GTP 對 F6P 抑制型態之分析。…...…....……….………………………....….40
圖 3-6 以不同 GTP 濃度和求得之 Km 作圖，並換算 GTP 的 Ki 值。….…………...41
圖 3-7 6P-GA 對 F6P 抑制型態之分析。...……………………………….…………..42
圖 3-8 以不同 6P-GA 濃度和求得之 Km 作圖，並換算 6P-GA 的 Ki 值。.………...43
圖 3-9 利用 Yonetani-Theorell 雙抑制測量法，對兩種濃度的 GTP 及不同濃度之 6P-GA 同時進行與 hPGI 結合活性之測試。………………………….…………………...44
圖 3-10 利用 Yonetani-Theorell 雙抑制測量法，對兩種濃度的 6P-GA 及不同濃度之 GTP 同時進行與 hPGI 結合活性之測試。..………………………….………………...45
圖 3-11 利用 UV-crosslinking 對 hPGI 點突變蛋白質與 [α-32P] GTP 結合分析。.....46
圖 3-12 利用 UV-crosslinking 對 ePGI 點突變蛋白質與 [α-32P] GTP 結合分析。.....47
圖 3-13 利用分子對接摸擬 hPGI 與 GTP 間結合作用位及作用關係圖之一。…….48
圖 3-14 利用分子對接摸擬 hPGI 與 GTP 間結合作用位及作用關係圖之二。…….49
圖 3-15 利用分子對接摸擬 hPGI 與 GTP 間結合作用位及作用關係圖之三。…….50
圖 3-16 利用分子對接摸擬 hPGI 與 GTP 間結合作用位及作用關係圖之四。…….51
圖 3-17 hPGI 晶體經 X ray 繞射分析之電子雲圖。…...…………………………….5
阿拉伯芥中四個調控花器老化凋落基因之功能性分析
No full text(1)FYF 基因為一抑制子去調控花朵老化及凋落。在本研究中，透過 microarray 實 驗找到三個受到 FYF 正調控的乙烯訊息因子 Ethylene-Responsive Factor (ERF) ，分 別是 FYF ACTIVATING FACTOR 2 (FAF2/ERF19) ，ERF87 及 ERF91。大量異位表現 FAF2 會延遲花朵老化及脫落 且增加花序上花朵數目 藉由共軛焦顯微鏡分析顯示、。， 大量表現 FAF2 使頂芽分生組織大小較野生型大 且花苞較同時期野生型多出一倍，， 大量表現 FAF2 促進頂芽分生組織活性決定性基因 WUS 表現，進而促進頂芽分生組 織活性，偵測細胞分裂素生合成及訊息傳遞路徑基因皆沒有明顯的差異，推測 FAF2 可能直接或藉由細胞分裂素以外路徑影響 WUS 基因的表現。利用 microarray 分析 FAF2 之下游調控路徑 結果顯示 FAF2 可能藉由抑制生長素生合成路徑基因 MYB51、，， CYP79B2 及 CYP71A13 使植株生長素含量降低，並經由抑制茉莉酸訊息傳遞路徑下 游 ORA47、RAP2.6、WRKY33、WRKY38、WRKY40、WRKY53 及 WRKY70 基因和同 為乙烯訊息傳遞及茉莉酸訊息傳遞路徑下游之 ORA59 進而調控花朵老化及頂芽分 生組織活性。 此外發現兩個同樣受 FYF 調控之下游基因 ERF87 及 ERF91，分析其各部位表 現量，顯示兩個基因皆在花朵發育早期表現量最高，而後隨花朵成熟表現量遞減， 且在 35S::FYF 穩定轉殖株中表現皆高於野生型阿拉伯芥，證實 ERF87 及 ERF91 可 受到 FYF 正調控 35S::ERF87 及 35S::ERF87-VP16 轉殖株出現小苗根短及藉由抑制。 FT 基因表現導致晚開花性狀，而 35S::ERF87-VP16 轉殖株觀察到經由促進 AS1 及 AS2 基因導致葉片上捲、透過促進 MYB75 及 GL3 基因造成轉殖株茸毛數量增加， 以及花萼瓣短小性狀，進一步分析結果推測 ERF87 藉由抑制 MYB21、MYB24 及 MYB57 進而抑制細胞分化及延展導致花萼瓣短小性狀 然而 35S::ERF87-SRDX 轉殖， 株則觀察到提早開花 小苗根長及花萼瓣變大性狀與 35S::ERF87 相反之突變性狀，、 根 據 以 上 結 果 認 為 ERF87 在 調 控 上 扮 演 活 化 子 角 色 。 另 外 ， 35S::ERF91 及 35S::ERF91-VP16 轉殖株藉由抑制 FT 基因表現導致延遲開花，而 35S::ERF91-SRDX 轉殖株則觀察到與 35S::ERF91 相反之早開花性狀，根據以上結果認為 ERF91 在調 控開花時間上扮演活化子角色。綜合上述結果，認為 ERF87 及 ERF91 確實受到 FYF 正調控，但其功能應是調控植株花器發育，並非調控花朵老化及花器脫落。
(2)自然環境中調控植物開花時間之機制有光週期、內生性、春化作用及吉貝素等 四個不同路徑 光週期路徑上 阿拉伯芥中由生理時鐘所調控之基因 CONSTANS (CO)。， 扮演調控開花的重要角色，CO 蛋白可以藉由活化下游基因 FLOWERING LOCUS T (FT) 表現促進開花。除了 CO 基因外，阿拉伯芥中還有 16 個 COL 基因同屬於 CO/COL 家族，而實驗室前人選殖了與 CO 屬於同一群組、蛋白質序列結構相同之 COL3 基因，分析其功能是否與 CO 相似，初步發現 COL3 與開花時間、花朵老化 及鹽耐受性調控有關，因此本研究將進一步分析 COL3 調控生長發育之機制。為分 析 COL3 表現位置，將 COL3 啟動子接上 GUS 進行轉殖，利用 GUS 染色及 real-time PCR 進行分析，結果顯示，GUS 蛋白表現在小苗之頂芽分生組織、子葉、真葉及根 部，花器部分則在後期成熟花之花萼、雄蕊及雌蕊頂端和基部，在前期未授粉花苞 表現量極低，隨花朵成熟表現量逐漸增加。為探討 COL3 之功能，觀察及分析轉殖 株性狀結果顯示，35S::COL3、35S::COL3-SRDX 及 35S::COL3-VP16 轉殖株均呈現 早開花性狀，利用 real-time PCR 偵測發現 COL3 藉由促進 FT 基因表現導致早開花 性狀。另外 35S::COL3 及 35S::COL3-SRDX 轉殖株出現提早花朵老化及花器脫落性 狀，提早花朵老化是經由促進乙烯訊息傳遞路徑下游 EDF1 及 EDF4 基因造成，利 用共軛焦顯微鏡及 real-time PCR 證實 COL3 透過促進離層相關基因 HSL2 及 HAESA 導致提早起始離層作用，造成提早花器脫落性狀，而 35S::COL3-SRDX 轉殖株出現 花萼瓣、雄蕊短小性狀是經由抑制茉莉酸訊息傳遞路徑下游 MYB21、MYB24 及 MYB57 影響花器發育所造成，根據上述結果推測 COL3 在調控花朵老化、花器脫落 及花器發育上扮演抑制子角色。(1)FYF has been thought to play a repressor role in controlling flower senescence/abscission. In this study, three Ethylene-Responsive Factor (ERF) genes, FYF ACTIVATING FACTOR 2 (FAF2/ERF19) , ERF87, ERF91, up-regulated by FYF were identified from microarray analysis. Ectopic expression of FAF2 delayed flower senescence/abscission and produced more flowers on inflorescence. Further confocol microscopy analysis found that the size of shoot apical meristem in 35S::FAF2 is bigger than that in wild-type and the number of flower buds is also more than that in wild-type. The production of more flowers in 35S::FAF2 was found due to the up-regulation of shoot apical meristem activity determined gene WUS, a similar effect caused by the activation of plant hormone cytokinin. Since the expression level of cytokinin synthesis and signal transduction related genes was not affected in 35S::FAF2 transgenic plants, we used microarray to analyze the putative downstream regulation pathway of FAF2. The result shows that FAF2 probably reduced auxin contents by inhibiting auxin biosynthesis genes MYB51, CYP79B2 and CYP71A13 to promote the expression of WUS gene. It also shows that FAF2 may regulate flower senescence by down-regulated jasmonate or ethylene signal transduction genes ORA47, ORA59, RAP2.6, WRKY33, WRKY38, WRKY40, WRKY53 and WRKY70. We proposed that FAF2 may regulate the WUS gene by down-regulating IAA biosynthesis or jasmonate signal transduction to affect shoot apical meristem activity. In this study, two other ERF genes, ERF87 and 91, up-regulated by FYF in 35S::FYF transgenic plant, were also characterized. The expression of ERF87 and 91 were decreased in mature flower and was up-regulated by 35S::FYF. Ectopic expression of ERF87 and ERF87-VP16 inhibited root development and delayed flowering by inhibiting FT expression. Ectopic expression of ERF87-VP16 also caused leaf incurvature by actitviting AS1 and AS2 gene expression and produced more trichome by promoting MYB75 and GL3. Furthermore, 35S::ERF87-VP16 also inhibited petal and sepal growth by down-regulating MYB21, MYB24 and MYB57 to inhibit cell division and expansion. By contrast, 35S::ERF87-SRDX showed the opposite phenotype to 35S::ERF87 by promoting flowering and the growth and the development of root and flower organs. These results indicate that ERF87 plays as an activator to regulate flower development. In addition, ectopic expression of ERF91 and ERF91-VP16 delayed flowering by suppressing FT expression whereas ERF91-SRDX promoted flowering. This data shows that ERF91 functions as an activator to regulate flowering. In summary, ERF87 and ERF91 were up-regulated by FYF to involve in flower development but not in senescence and abscission.
Flowering is controlled by several environmental signals such as photoperiodic, autonomous, vernalization and GA pathway. In the photoperiodic pathway, the circadian-regulated gene CONSTANS (CO) plays an important role to regulate the flowering in Arabidopsis, CO protein is able to promote the flowering through the activation of the downstream gene FT. In addition to CO gene, there are 16 other COLs genes belonging to CO/COL family. COL3 which belongs to the same subgroup with CO has been cloned for functional analysis. It has been found that COL3 could involve in the regulation of flowering time, flower senescence/abscission and salt tolerance. This study further analyzed the mechanisms of COL3 in regulating plant growth development. COL3 expressed in shoot apical meristem, cotelodon, true leave and root of 7-day and 14-day-old seedlings. The expression of COL3 was higer in mature flower than in flower buds. In flowers, COL3 expressed in sepals and filament of mature flower as well as in silique. Ectopic expression of COL3, COL3-SRDX, COL3-VP16 promoted flowering by activating FT gene. Ectopic expression of COL3 and COL3-SRDX also caused early flower senescence by activating ethylene signal transduction genes EDF1 and EDF4, and also promoted abscission by activating abscission-associated genes HSL and HAESA. Furthermore, ectopic expression COL3-SRDX produced short flower organs by suppressing jasmonate signal transduction genes MYB21, MYB24 and MYB57. These results suggest a possible repressor role for COL3 in controlling flower senescence and abscission.第一章 調控花朵老化凋落之 ERF 基因之功能性分析
摘要…..1
ABSTRACT…..2
前言…..3
材料方法…..6
結果…..14
一、大量異位表現 FAF2 增加阿拉伯芥花朵發育數目性狀之分析…..14
二、藉由 cDNA 微陣列晶片分析 FAF2 之下游調控基因…..15
三、阿拉伯芥中 ERF87 及 ERF91 之序列分析…..16
四、野生型阿拉伯芥各部位及花朵各時期中 ERF87 及 ERF91 基因表現 量之偵測…..16
五、35S::FYF 之 T4 代穩定轉殖株中 ERF87 及 ERF91 基因表現量之偵測…..17
六、阿拉伯芥中 ERF87 及 ERF91 啟動子之選殖和 ERF87P::GUS 及 ERF91P::GUS之構築…..17
七、阿拉伯芥中 ERF87 及 ERF91 基因之分子選殖及重組載體構築…..18
八、大量異位表現 ERF87、ERF87-SRDX 及 ERF87-VP16 在阿拉伯芥中造成開花時間差異之分析…..19
九、大量異位表現 ERF87 造成根短性狀之分析…..20
十、大量異位表現 ERF87-VP16、ERF87-SRDX 造成花萼瓣大小異常性狀之分析…..21
十一、大量異位表現 ERF87-VP16 造成上捲葉及茸毛 (trichome) 數量較多性狀之分析…..21
十二、大量異位表現 ERF91 造成晚開花性狀之分析…..22
討論…..24
參考文獻…..28
圖表附錄…..31
第二章 調控花朵老化凋落之 COL 基因之功能性分析
摘要…..73
ABSTRACT…..74
前言…..75
材料方法…..78
結果…..82
一、 阿拉伯芥中 COL3 啟動子之選殖和 COL3p::GUS 之構築…..82
二、 藉由 GUS 染色分析 COL3 於阿拉伯芥中不同生長時期和部位之表現情形…..82
三、 大量異位表現 COL3 造成早開花性狀之分析…..83
四、 大量異位表現 COL3 造成花器提早老化性狀之分析…..83
五、 大量異位表現 COL3 造成花器提早脫落性狀之分析…..84
六、 大量異位表現 COL3-SRDX 造成花萼、花瓣及雄蕊短小性狀之
分析....85
討論…..86
參考文獻…..88
圖表附錄…..9
Preparation of micro hydrogel beads composed of fermented soy milk and okara and the evaluation its releasing properties
No full textRecently, health conscious of national people raising up results in output value of functional food increasing. The output value is about 85,000 million NT dollars in 2014. The cereals are the largest source of functional foods materials. Soybean is a regular consumption in Taiwan and it is rich in nutrients and functional compounds. The soybean amount of input in Taiwan is 173,469 tons in a whole year. Besides, soybean is a popular food because of unique flavor. Okara is a main by-product during the processing of soybean products. The annual output of okara was over 366,019 tons in Taiwan. It is abundant in moisture, protein and dietary fiber content. Recently, probiotics and related products are popular topic. Therefore, this research study on fermentation liquid, okara and micro hydrogel beads.
The results show that the best ratio of soybean milk and okara, components of fermentation liquid, is 60:1 named SM60O1. After 24 hours fermentation, the microbial counts, pH value, total phenolic compounds content, total flavonoid content, carbohydrate content and titratable acidity are 8.76 log CFU/ml, 4.15, 0.59 mg/g dw, 0.24 mg/g dw, 1.64 mg/ml and 0.41%. Particle size results of okara fiber treated by different extents of micronized processing are 416.48, 304.92, 185.51, 48.48 and 20.30 μm. Moreover, the 20-mesh group, mean particle size is 416.48μm, has the best water-holding capacity, swelling capacity and oil-holding capacity, 9.12, 16.46 and 3.51 ml/g respectively. The complex hydrogel bead is made by 7.5% pectin and 1 % arabic gum and the ratio of them is 3:2. It is the best prepared condition and named P3A2. The moisture content, swelling capacity, hardness and enthalpy analysis of this complex hydrogel bead are 96.85%, 230.13%, 4865.6 g and 98.4 J/g. The functional hydrogel bead named SM1G2, means the ratio of soybean milk and gel is 1:2, has the best strength of bonding structure. The moisture content, swelling capacity, hardness and enthalpy analysis are 99.72%, 180.36%, 3500.67 g and 33.23 J/g. Moreover, the probiotic survival rate of SM1G2 in the stimulate gastric juice, pH value are 1.2 and 2.0, are 57.29 and 95.37%. And the microbial counts of releasing test in the intestinal liquid, after stimulate gastric juice, is 7.31 log CFU/ml. In this study, the best condition is SM1G2 functional hydrogel bead. The best concentration and ratio of mixed gel are 1 % and 3:2 of arabic gum and pectin.近年來國人健康意識抬頭 於 2014 年健康食品市場產值上看 850，億元新台幣 其原料來源廣泛 以植物的使用位居首位 黃豆 (soybean)。為我國常食用之豆類原料，含有豐富之營養物質及機能性成分，統計資料顯示黃豆粉及其細粒總量全年之進口量達 1904,829 公噸，由於其具有獨特風味廣受國人喜愛，在豆類製品的加工過程中，豆渣(okara) 為主要副產物，我國每年產生的豆渣數量高達 58 公噸，其水分及蛋白質含量高，亦可作為膳食纖維的良好來源。益生菌及其相關製品亦為近年來保健食品之熱門主題，因此，本實驗探討豆渣之機能性成分分析及理化特性評估，並探討以果膠及阿拉伯膠之混合膠體製備具機能性之發酵豆乳 complex 式膠球造粒品質及益生菌之存活率，最後評估其體外模擬釋放試驗。
本實驗結果如下：豆漿與豆渣混合共同發酵之最佳比例為SM60O1，其發酵 24 hr 後之菌數達 8.76 log CFU/ml，pH 值為 4.15，總多酚和類黃酮分別為 0.59 mg gallic acid eq./ g dw 和 0.24 mgquercetin acid eq./ g dw，總醣及可滴定酸為 1.64 mg/ml 和 0.41%。不同粒徑之豆渣纖維其粒徑分佈分別為 416.48、304.92、185.51、48.48及 20.30 μm，其中以 416.48 μm (20 mesh) 有最佳的保水力、膨潤力和保油力，分別為 9.12、16.46 及 3.51 ml/g。果膠與阿拉伯膠混合比例以 1% P3A2 complex 式膠球為最適製備載體條件 其含水率及膨潤，力分別為 96.85 及 230.13%，硬度測試 4865.6 g，熱焓分析 98.4 J/g。發酵豆乳機能性膠球以 SM1G2 有最佳的鍵結強度，含水率及膨潤力分別為 99.72 及 180.36%，硬度測試 3500.67 g，熱焓分析 33.23 J/g，於 pH 1.2 及 2.0 的模擬胃液中，SM1G2 之菌體耐受性較佳，其存活率分別為 57.29 和 95.37%，在胃腸連續釋放試驗中，菌體之釋放量達7.31 log CFU/ml。中文摘要..............I
Abstract ..............II
目次..............IV
圖表索引..............XI
表次..............XI
圖次..............XIII
壹、引言..............1
貳、文獻回顧..............3
一、黃豆 ( soybean ) 之簡介、組成分及機能成分 ..............3
(一)黃豆簡介 ..............3
(二)黃豆之一般組成分..............6
(三)黃豆之機能性成分..............6
1.黃豆胜肽（soy peptide）..............6
2.黃豆固醇（soy sterols） ..............10
3.卵磷脂 (lecithin) ..............10
4.大豆異黃酮 (isoflavone) ..............11
5.豆渣纖維 (okara) ..............12
二、豆渣 (Okara) ..............15
三、益生菌 (probiotics) ..............18
(一)益生菌的起始 ..............18
(二)益生菌的定義..............18
(三)益生菌的分類 ..............19
(四)益生菌之特性..............23
(五)益生菌的保健功效..............26
1.改善腸道菌相： ..............26
2.增進免疫力：..............26
3.改善腸道不適症狀： ..............26
4.改善乳糖不耐症 ..............27
5.降低膽固醇..............27
(六)益生菌於食品工業上的應用..............27
四、膳食纖維 (dietary fiber) ..............30
五、微膠囊包埋與造粒技術 ..............31
(一)微膠囊之簡介..............31
(二)壁材 ..............32
1.果膠 (pectin) ..............38
2.阿拉伯膠..............40
3.幾丁聚醣 (chitosan) ..............50
(三)微膠囊的製備方法 ..............54
1.噴霧乾燥法 ..............54
2.銳孔---凝固浴法 ..............57
3.乳化交聯法..............57
4.相分離法 ..............57
六、微膠囊之控制釋放技術..............59
1.擴散控制釋放系統（diffusion controlled release） ...........59
2.膨溶控制釋放系統 (swelling controlled release system) ...60
3.侵蝕或化學反應控制系統 (erosion or chemical reaction controlled system) ..............60
参、實驗目的..............63
肆、實驗架構..............64
伍、材料與方法 ..............65
一、實驗材料 ..............65
(一)豆漿 (soybean milk)..............65
(二)豆渣 (okara) ..............65
(三)實驗菌株 ..............65
1.菌種： ..............65
2.菌株活化與保存： ..............65
(四)培養基與藥品 ..............66
(五)發酵豆乳樣品之製備..............66
1.菌液活化..............66
2.發酵豆乳製備 ..............66
二、實驗方法 ..............67
(一) 基本成分分析..............67
1.水分（moisture） ..............67
2.灰分（ash） ..............67
3.粗蛋白質（crude protein） ..............67
4.粗脂肪（crude fat） ..............68
5.粗纖維（crude fiber） ..............68
6.碳水化合物（carbohydrate） ..............69
(二)發酵液品質評估..............69
1.抗氧化能試驗..............69
2.菌數測定 (The measure for bacterial count)..............70
3.總醣量（total carbohydrates content）之測定 ....................70
4.可滴定酸測定(titratable acidity) ..............71
5.色澤分析 (color analysis..............71
6.濁度 (turbidity) ..............72
7.黏度測定 (viscosity) ..............72
8. pH 值..............72
9.糖度(°Brix)測定..............72
(三)豆渣之理化特性評估..............72
1.粒徑大小 (particle size) ..............72
2.色澤分析 (color analysis..............73
3.掃描式電子顯微鏡 (scanning electron microscopy, SEM) .73
4.解剖顯微鏡 (stereomicroscope)..............73
5. X-射線繞射分析 (X-ray diffraction analysis) ..............73
6.膨潤力 (swelling capacity) ..............74
7.保水力 (water-holding capacity) ..............74
8.保油力 (oil-holding capacity) ..............74
9.體積密度 (bulk density) ..............75
10.陽離子交換力 (cation-exchange capacity) ..............75
(四)豆渣之機能性成分分析..............75
1. DPPH 清除自由基能力（DPPH scavenging activity）測定..............75
2.總多酚 (total phenolic) 含量測定..............76
3.類黃酮 (total flavonoid) 含量分析 ..............76
4.膳食纖維 (dietary fiber) ..............76
(五)膠球製備方法..............78
(六)造粒品質分析 ..............79
1.掃描式電子顯微鏡(scanning electron microscopy, SEM) ...79
2.解剖顯微鏡 (stereomicroscope)..............79
3.粒徑大小（particle size） ..............79
4.含水率 (moisture content) ..............80
5.膨潤力 (swelling capacity) ..............80
6.硬度(hardness) ..............80
7.示差熱掃描分析 (DSC) ..............83
8.鈣離子濃度測定 (determination of calcium ion concentration) ..............83
9.黏度測定 (viscosity) ..............83
10.包覆率 (encapsulation yield..............83
(七)膠球之溶離試驗 ..............84
1.模擬胃液配製..............84
2.模擬腸液配製 ..............84
3.乳酸菌之胃液耐受性 ..............84
4.模擬腸胃道連續釋放試驗..............85
(八) 統計分析 (statistical analysis) ..............85
陸、代號說明..............86
柒、結果與討論 ..............87
第一章：含豆渣發酵豆乳之製備及其機能性成分分析 ..............87
一、豆漿之基本成分分析 ..............88
二、發酵豆乳之品質評估 ..............88
(一)發酵期間菌數變化及 pH 值之測定..............88
(二)機能性成分分析 ..............89
(三)總醣含量測定及糖度測定..............89
(四)可滴定酸含量測定 ..............90
(五)黏度測定..............101
(六)濁度安定性變化..............101
(七)色澤變化..............102
三、小結..............106
第二章：豆渣膳食纖維－機能性成分及其理化特性評估 ........107
一、粒徑分佈 ..............108
二、外觀及微細構造 ..............108
三、色澤分析..............108
四、X 光繞射分析 (X-ray) ..............109
五、機能性成分含量分析 ..............109
(一)膳食纖維含量測定..............109
(二)總多酚及類黃酮含量測定..............110
六、理化特性評估..............119
(一)體積密度 (bulk density) ..............119
(二)保水力 (water-holding capacity) 和膨潤力 (swelling capacity) ..............119
(三)保油力 (oil-holding capacity) ..............120
(四)陽離子交換力(cation-exchange capacity) ..............120
七、小結..............126
第三章：果膠-阿拉伯膠乳化微晶球粒子之製備及其品質評估..............127
一、製備不同條件之膠球外觀 ..............128
二、製備不同條件之膠球鍵結強度 ..............133
(一)製備不同條件之膠球含水率..............133
(二)製備不同條件之膠球膨潤力..............133
(三)製備不同條件之膠球硬度..............134
(四)製備不同條件之膠球的熱焓分析..............135
(五)不同條件製備之膠球微細構造..............138
(六)製備不同條件之膠球 X 光繞射分析..............138
三、經二次包覆之膠球外觀..............143
四、經二次包覆之膠球鍵結強度..............149
(一)二次包覆膠球之含水率..............149
(二)二次包覆膠球之膨潤力..............149
(三)二次包覆膠球之硬度測試..............152
(四)二次包覆膠球之熱焓分析..............152
五、小結..............157
第四章：果膠-阿拉伯膠乳化微晶球粒子模擬腸胃道試驗及其釋控能力評估..............160
一、不同處理組別膠球之外觀及粒徑大小 ..............161
二、不同處理組別膠球之鍵結強度 ..............161
(一)不同處理組別之膠球含水率及膨潤力 ..............161
(二)不同處理組別之膠球硬度測試及熱焓分析 ..............161
(三)不同處理組別之膠球微細構造..............162
三、不同處理組別膠球之釋放特性 ..............168
(一)膠球之包覆率..............168
(二)模擬胃液中膠球之外觀及微細構造..............168
(三)模擬胃液中之乳酸菌耐受性..............169
(四)模擬腸液中膠球之外觀..............169
(五)胃腸中膠球之連續釋放能力評估..............170
四、小結..............184
捌、結論..............185
玖、參考文獻..............18
Apoptotic effect of the extract from Antrodia salmonea mycelim and solid-state fermented products on cancer cells
No full textAntrodia salmonea, which grows on the empty rotten trunk of Cunninghamia konishii in Taiwan, is a new species of the genus Antrodia as a basidiomycete idenfied in 2004. Many studies have suggested that the extract of A. salmonea, both fruiting body and mycelium, shows the abilities of anti-oxidative, anti-atherosclerotic, anti-inflammatory, and also have antioxidative ability in human leukocytes. We investigates the apoptotic cells of the 70% ethanol extract from A. salmonea mycelium, A. salmonea fermented buckwheat (ASFB) and A. salmonea fermented oat (ASFO) in Sk-Hep1cells and Caco2 cells.
The physiological activity in ergothioneine, total phenol and flavonoids were studied. The total ergothioneine cotent of A. salmonea mycelium (168.06 µg/g) was higher than ASFO (138.70µg/g) and ASFB (118.64µg/g). Both the total phenol and tota flavonoids of A. salmonea mycelium solid state fermented products (ASF and ASFB) were higher than A. salmonea mycelium. Contents of total phenol was ASFB (37.58 µg/mL)、ASFO (24.09 µg/mL) and A. salmonea mycelium (13.49 µg/mL). Contents of tota flavonoids.was ASFB (1.37 µg/mL)、ASFO (0.36 µg/mL) and A. salmonea mycelium (0.11 µg/mL)。
We used MTT assay to measured that the cell viability of Sk-Hep1 cells and Caco2 cells, and the results exhibited dose- and time- dependent response, but no effect on 293T cells. The main mechanism of the apoptotic effect were included to arrest G1/G0 phase, subG1 phase. While the proportion apoptotic cells of 293T cells have concentration- and time- dependent response, but showed no significantly different in 24h. After 48h incubation.only the apoptotic cells of ASFB (250 µg/mL) was 3.83%, which have significantly different compare with control. We evalutated the mitochondrial membrane potential (△Ψm) by JC-1 stain. Results reveled that 70% ethanol extracts from A. salmonea mycelia, ASFB and ASFO caused a significantly loss of mitochondrial membrane potential (△Ψm) at 250 µg/mL. The results exhibited the up-regulated expression of Caspase-3,8,9 in 70% ethanol extract from A. salmonea mycelia , ASFB and ASFO.
In conclution, we demonstrated that 70% ethanol extract from A. salmonea mycelia and A. salmonea fermented products (ASFB and ASFO) might suppress the proliferation in Sk-Hep1 cells and Caco2 cells by inducing apoptosis. A. salmonea mycelia、ASFB and ASFO triggered apoptosis activities by arresting G1/G0 phase, subG1 phase, a significantly loss of mitochondrial membrane potential, and promoting activities of caspase-3, caspase-8 and caspase-9.. Thses data indicated that the apoptosis-inducing effects of A. salmonea mycelia and A. salmonea solid-state fermented products (ASFB, ASFO) might have the new choice as a potential chemotherapeutic agents.香杉芝 (Antrodia salmonea) 為寄生於香杉木上的台灣特有真菌，於 2004 年被鑑定出為薄孔菌屬之新種。香杉芝子實體與菌絲體萃取物具有抗氧化、抗動脈硬化、和抗發炎等功效，且對人類白血球細胞具有抗氧化作用。本實驗之研究目的為探討香杉芝菌絲體及香杉芝穀物 (蕎麥、燕麥) 固態發酵產品之 70% 乙醇萃取物，誘導大腸癌細胞與肝癌細胞凋亡的特性。
在生理活性物質方面，香杉芝菌絲體之麥角硫因含量為 168.06 µg/g 高於香杉芝燕麥 (ASFO) 138.70 µg/g 和香杉芝蕎麥 (ASFB) 118.64 µg/g。香杉芝固態發酵產品總多酚及類黃酮含量均高於香杉芝菌絲體。總多酚類含量依序為 ASFB (37.58 µg/mL)、ASFO (24.09 µg/mL) 及香杉芝菌絲體 (13.49 µg/mL)；類黃酮含量依序為 ASFB (1.37 µg/mL)、ASFO (0.36 µg/mL) 及香杉芝菌絲體 (0.11 µg/mL)。
由流式細胞儀分析細胞週期分布與細胞凋亡結果可知，人類大腸癌細胞 (Caco2)、人類肝癌細胞 (Sk-Hep1) 細胞增生能力，會隨著萃取物濃度升高及時間增加而被抑制；此外，萃取物不會對人類正常腎臟上皮細胞 (293T) 細胞株有抑制生長之效果。
接著探討誘導細胞凋亡之機制。經流式細胞儀偵測其細胞週期，實驗結果發現香杉芝菌絲體、ASFB、ASFO 可透過使細胞之 G0/G1 期上升和增加 subG1 來提升細胞凋亡比率；然而， 293T在細胞凋亡比率雖然會隨著作用濃度上升，但於 24 小時與控制組沒有顯著差異。僅 ASFB 組別作用 48 小時，濃度為 250 µg/mL 時，細胞凋亡比率為 3.83%，與控制組有顯著差異。另由 JC-1 之方法測定粒線體膜電位變化 (△Ψm），結果發現濃度為 250 µg/mL 時，粒線體膜電位有明顯下降的趨勢。此外，在細胞凋亡的訊息傳導路徑中，發現香杉芝菌絲體、 ASFB、 ASFO 均可使 Caspase-3,8,9活性顯著增加。
綜上所述，香杉芝菌絲體、ASFB 及 ASFO 具有誘導人類大腸癌細胞 (Caco2)、人類肝癌細胞 (Sk-Hep1) 凋亡的能力，推測其誘導凋亡路徑可由上升 G0/G1 期、subG1 期、降低粒線體膜電位及增加 Caspase-3,8,9 活性啟動細胞內一系列的凋亡。由本研究可知，香杉芝穀物之固態發酵產品具有誘導肝癌細胞及大腸癌細胞凋亡的潛力，可提供在抗癌應用上新的選擇。表次 viii
圖次 ix
縮寫表 xv
前言 1
文獻回顧 3
一、香杉芝 Antrodia salmonea 3
二、固態發酵之介紹 3
三、癌症 6
(一) 肝癌 6
(二) 大腸癌 8
四、抗癌藥物 9
(一) 蕾莎瓦 Sorafenib 9
(二) 希樂葆 Celecoxib 9
五、細胞週期 12
六、細胞凋亡 14
(一) 凋亡路徑 14
(二) Caspase 3, -8, -9 16
研究動機 17
實驗架構 18
材料與方法 19
一、實驗材料 19
(一) 香杉芝菌種 19
(二) 細胞株 19
(三) 試藥 19
二、實驗方法 20
(一) 香杉芝菌絲體粉末製備 20
(二) 香杉芝固態發酵產品粉末製備 20
(三) 乙醇萃取物之製備 20
(四) 生理活性物質測定 21
(五) 細胞株培養 22
(六) 細胞存活率試驗 23
(七) 細胞週期 (Cell cycle) 23
(八) 細胞凋亡分析 24
(九) 粒線體膜電位測定 24
(十) Caspase活性分析 25
(十一) 統計分析 25
結果與討論 26
一、固態發酵產品麥角固醇含量測定 26
二、樣品萃取率與生理活性物質分析 26
三、細胞存活率試驗 31
四、細胞週期分析 47
五、細胞凋亡試驗 74
六、粒線體膜電位分析 102
七、Caspase-3, -8, -9 活性分析 112
結論 141
參考文獻 14
Effect of high-advanced glycation end product (AGE) diet on oxidative damage of testes, kidney and pancreas in normal and diabetic rodents,and the intervention effect of silymarin
No full textAdvanced glycation end products (AGEs) are produced from the Maillard reaction and present in food and biological systems. The present studies show that high levels of circulating AGEs are associated with diabetic complications, such as nephropathy, retinopathy and atherosclerosis.It is worth noting that male diabetic patients may also have reproductive dysfunction, and this dysfunction may result from the oxidative stress induced by AGE-mediated the receptor for AGEs (RAGE) activation. Sustained intake of food rich in AGEs may lead to an increase in endogenous AGEs and chronic oxidative damage. Taken together, it is interesting to clarify the effect of AGE diet on function of testes, kidney and pancreas in normal and diabetic anamals. Silymarin is a flavonoid with hepatoprotective characteristics and powerful antioxidant activity.Therefore, the other section of study was further investigated to clarify the biological actions of silymarin in animals fed an AGE diet. Our study is divided into two sections as follows:
In part 1, we assessed the effect of different formula of AGE diet on testes, kidney and pancreas in male BALB/c mice and Sprague-Dawley rats, respectively, and the effect of the biological actions of silymarin. The results showed that AGE diet may lead to accumulation of AGEs in vivo, resulting in oxidative damage of testes and epididymis as well as a decrease
in sperm count and motility. To investigate the intervention effect of silymarin, we found that silymarin had potential for prevention of AGE
diet-induced oxidative damage. Based on the results of first part, AGE diet
was shown to cause accumulation of AGEs. Because the circulating AGEs
are closely related to the progression of diabetic complications, it is
speculated that diabetic damage of testes and epididymis may be augmented by AGE diet. In part 2, we investigated the effect of AGE diet on STZ-induced type 1 diabetes mellitus (DM) SD rat as well as the intervention effect of silymarin. The results showed that DM-induced oxidative damage led to a decrease of sperm count and motility, and AGE diet may aggravate related lesions, such as an increase of abnormal sperm rate. However, silymarin elevated activity of antioxidant enzymes of testes and ameliorated the lipid peroxidation in pancreas. Silymarin showed the potential for improving DM-induced oxidative damage in vivo; however, it did not significantly improve the STZ and AGE diet-induced decline of sperm count and diffuse spermatic degeneration/necrosis of epididymis. We speculate that the related damage of animals may be difficult to restore; thus, we cannot observe the expected results in related analyses.
In conclusion, diversity and preparation method of diet, experimental models, and animal strain might be the reasons why partial results are inconsistent. Based on all results, we summerize that AGE diet-induced oxidative stress are closely related to abnormalities of sperm in rodents. Additionally, administration of silymarin indeed significantly promoted antioxidant actions within testes, kidney and pancreas in rodents, suggesting that silymarin has potential to inhibit the generation of lipid peroxides and improvement of oxidative damage in vivo.高度醣化終產物(advanced glycation end products, AGEs)為梅納反應產物，其可於食品及生物體內生成。研究顯示，糖尿病患體內高AGEs濃度與糖尿病併發症 (例如：腎病變、視網膜病變及動脈粥狀硬化)之發生有密切相關。值得注意的是，男性糖尿病患者也可能出現生殖功能異常之現象，究其因可能為AGEs與其細胞專一接受器(the receptor for AGEs, RAGE)交互作用所衍生之氧化壓力所致。由於長期攝食含高量AGEs之食物亦可能會導致體內AGEs升高並造成慢性氧化傷害，因此本研究擬釐清AGE飲食對正常及糖尿病個體內睪丸、腎臟及胰臟功能之影響。水飛薊素(silymarin,SM)為一種具護肝特性與強抗氧化能力之類黃酮(flavonoid)化合物。基於此，本研究亦同時評估水飛薊素對餵食AGE飼料動物之作用。本研究分為兩部分，相關研究結果分述如下：
第一部分設計不同配方之AGE飲食，探討其對雄性BALB/c小鼠及Sprague-Dawley(SD)大鼠睪丸、腎臟及胰臟之影響，同時探討介入水飛薊素之生理效應。實驗結果顯示，由AGE飲食型態造成之體內AGEs堆積可能會引起睪丸、副睪產生氧化傷害，進一步造成精子總數及活動力下降。此外，水飛薊素具有改善AGE飲食引起氧化
傷害之潛力。
由前部分研究結果可知，AGE飲食會造成體內AGEs堆積，由於體內AGEs含量與糖尿病併發症有絕對相關性，故AGE飲食極可能加劇糖尿病併發症及其引起之睪丸、副睪損傷。因此，第二部分之研究即探討AGE飲食對Streptozotocin (STZ)誘導第一型糖尿病SD雄鼠之影響，同時觀測介入水飛薊素之生理效應。結果證實，糖尿病引起之氧化傷害會造成精子總數及活動力下降，且AGE飲食型態可能會使精子異常率提升。然而，水飛薊素可提升睪丸之抗氧化酵素活性，及減緩胰臟脂質過氧化之現象，具有改善糖尿病所造成體內氧化傷害之潛力。另外，水飛薊素對於糖尿病及AGE飲食引起之精子總數下降、副睪精子變性壞死之現象，無明顯改善，推測可能為動物體遭受之傷害已達難以恢復之程度，故於分析數據上無法觀測到預期之結果。
總結以上，飼料種類及製備方法、實驗期長短、實驗模式、以及老鼠品系可能是造成本研究部分結果不一致之原因。儘管如此，本研究成果發現，AGE飲食引起之氧化壓力為生物體精子異常之重要原因。另外，服用水飛薊素可提升生物體內睪丸、腎臟及胰臟之抗氧化能力、抑制脂質過氧化物生成，顯示其具有改善氧化傷害之潛力。全文摘要 i
Abstract iii
縮寫表 i
前言 1
第一章文獻整理 3
壹、糖尿病(Diabetesmellitus,DM) 4
一、糖尿病簡介 4
二、糖尿病主要類型 4
三、糖尿病併發症 6
四、高血糖誘發併發症之相關機轉 8
五、糖尿病之動物模式 10
貳、高度醣化終產物(Advancedglycationendproducts,AGEs) 21
一、高度醣化終產物之生成 21
二、高度醣化終產物之種類 22
三、高度醣化終產物與疾病 23
參、男性生殖系統與不孕症(infertility) 28
一、男性生殖系統之簡介 28
二、不孕症 28
肆、水飛薊素(Silymarin) 34
一、水飛薊 34
二、水飛薊素之特性 34
三、水飛薊素之生理活性 34
伍、研究目的 39
陸、研究架構 40
第二章、水飛薊素對高度醣化終產物(AGE)飲食之雄鼠睪丸、腎臟及胰臟之影響 42
摘要 43
Abstract 45
前言 47
實驗架構 48
材料方法 49
結果 61
1.水飛薊素對AGE飲食之雄性BALB/c小鼠睪丸及腎臟之影響 61
1.1.雄性BALB/c小鼠飲食中AGE含量之分析 61
1.2.AGE飲食對雄性BALB/c小鼠攝食量之影響及AGE攝取量之分析 61
1.3.水飛薊素對AGE飲食之雄性BALB/c小鼠血糖之影響 61
1.4.水飛薊素對AGE飲食之雄性BALB/c小鼠體重、臟器重量及大小之影響 62
1.5.水飛薊素對AGE飲食之雄性BALB/c小鼠精子特性之評估 62
1.6.水飛薊素對AGE飲食之雄性BALB/c小鼠血清、睪丸及腎臟組織中脂質過氧化之影響 62
1.7.水飛薊素對AGE飲食之雄性BALB/c小鼠睪丸及腎臟組織中抗氧化酵素之影響 63
1.8.水飛薊素對AGE飲食之雄性BALB/c小鼠血清睪固酮濃度之影響 63
1.9.水飛薊素對AGE飲食之雄性BALB/c小鼠血清、睪丸及腎臟組織中AGE含量之影響 64
1.10.組織病理分析(H&E染色) 64
1.11.水飛薊素對AGE飲食之雄性BALB/c小鼠睪丸及腎臟之蛋白質表現 65
2.水飛薊素對AGE飲食之雄性SD大鼠睪丸及胰臟之影響 67
2.1.雄性SD大鼠飲食中AGE含量之分析 67
2.2.水飛薊素對AGE飲食之雄性SD大鼠體重變化之影響 67
2.3.AGE飲食對雄性SD大鼠攝食量之影響及AGE、SM攝取量之分析 67
2.4.水飛薊素對AGE飲食之雄性SD大鼠血糖之影響 68
2.5.水飛薊素對AGE飲食之雄性SD大鼠臟器重量及大小之影響 68
2.6.水飛薊素對AGE飲食之雄性SD大鼠精子特性之評估 68
2.7.水飛薊素對AGE飲食之雄性SD大鼠血清、睪丸及胰臟組織中脂質過氧化之影響 69
2.8.水飛薊素對AGE飲食之雄性SD大鼠睪丸及胰臟組織中抗氧化酵素之影響 69
2.9.水飛薊素對AGE飲食之雄性SD大鼠血清睪固酮及胰島素濃度之影響 70
2.10.水飛薊素對AGE飲食之雄性SD大鼠血清生化參數之影響 70
2.11.水飛薊素對AGE飲食之雄性SD大鼠血清、睪丸及胰臟組織中AGE含量之影響 70
2.12.組織病理分析(H&E染色) 70
2.13.水飛薊素對AGE飲食之SD大鼠睪丸蛋白質表現之影響 71
討論 72
第三章、AGE飲食對STZ誘導糖尿病SD雄鼠之影響暨介入水飛薊素之功效 114
摘要 115
Abstract 116
前言 118
實驗架構 119
材料方法 120
結果 130
一、糖尿病大鼠飲食中AGE含量之分析 130
二、水飛薊素對AGE飲食之糖尿病大鼠體重變化之影響 130
三、水飛薊素對AGE飲食之糖尿病大鼠攝食量、AGE及SM攝取量之影響 130
四、水飛薊素對AGE飲食之糖尿病大鼠血糖之影響 130
五、水飛薊素對AGE飲食之糖尿病大鼠臟器重量及大小之影響 131
六、水飛薊素對AGE飲食之糖尿病大鼠精子特性之評估 131
七、水飛薊素對AGE飲食之糖尿病大鼠精子、血清、睪丸及腎臟組織中脂質過氧化之影響 131
八、水飛薊素對AGE飲食之糖尿病大鼠睪丸及胰臟組織中抗氧化酵素之影響 132
九、水飛薊素對AGE飲食之糖尿病大鼠血清睪固酮及胰島素濃度之影響 132
十、水飛薊素對AGE飲食之糖尿病大鼠血清生化參數之影響 132
十一、水飛薊素對AGE飲食之糖尿病大鼠血清、睪丸及胰臟組織中AGE含量之影響 133
十二、組織病理分析(H&E染色) 133
十三、睪丸蛋白質表現量 134
討論 135
總結論 160
參考文獻 16
Development of a lactic acid-fermented fruit juice
No full textLactic acid-fermented products which have their own fermentation process have been popular across the globe for a long time. The objective of this study is to develop a lactic acid-fermented fruit juice and to evaluate its potential for the improvement of intestinal health. This research was divided into three parts; firstly, lactic acid fermentation was compared with mixed fermentation. After the comparison trial, the better method was adapted for the subsequent fermentations. Secondly, the experiments were conducted by adding different fruit combinations, pectinase (PE) and isomalto-oligosaccharides (IMO). Finally, the fermentation model was adapted based on our experimental results. The optimal formula of fermented products was chosen and assessed by both in vitro and in vivo methods.
In the comparison of lactic acid fermentation and mixed fermentation, the lactic acid fermentation was found to have better effect than the mixed fermentation. The trial also showed that concentrated juice could not replace fresh pulp on the lactic acid fermentation. Furthermore strong flavor fruits could not influence the flavor of the final product. However it was found that adding PE could enhance the yield of lactic acid-fermented fruit juice. But, the addition of IMO had no prebiotic effect on the lactic acid-fermented fruit juice. According to the above experimental results, the fermentation model was established. Three formulas were designed for the preparation of the lactic acid-fermented fruit juice, and then the optimal one was selected for physiological assessments. The results of the in vitro assay showed that the sample did not have obvious prebiotic effect. Meanwhile, the formula was used for in vitro assay. The results indicated that the consumption of the lactic acid-fermented fruit juice could significantly (p<0.05) increase the number of total lactic acid bacteria in the intestine of mice. This study has developed a lactic acid-fermented fruit juice which could improve intestinal micro flora and promote intestinal health.乳酸發酵液流行已久，但各產品的發酵方式不一，風味也不盡相 同。本研究欲探討天然果汁乳酸發酵之開發，評估是否具有改善腸道 健康之潛力。實驗主要共分為三個部分，先會比較直接乳酸發酵與混 合式發酵之模式，選擇較適合的模式進行後續發酵試驗，然後探討不 同原料組合、果膠酶(pectinase)與異麥芽寡糖(isomalto-oligosaccharides) 對發酵果汁之影響，最後以前兩部分建立之發酵模式進行乳酸發酵果 汁的配方比較，選取最適配方後進行體外及體內的試驗評估。
首先於直接乳酸發酵與混合式發酵法的比較中，直接乳酸發酵在 發酵試驗結果中較混合式發酵理想。接著，於探討發酵條件階段裡， 觀察到濃縮果汁無法取代新鮮果肉作為乳酸發酵的材料、添加風味較 重的水果對發酵液的味道影響有限、加入果膠酶可使發酵液產率提升 及異麥芽寡糖的乳酸菌助生性效果不理想等發現。最後，設計 3 種不 同原料配方，以本研究建立之乳酸發酵流程發酵並選出較佳配方進行 生理活性評估。體外試驗顯示樣品無明顯助生性，但在體內試驗中， 結果顯示乳酸發酵果汁能顯著(p<0.05)提升小鼠腸道內總乳酸菌數，有 助腸道菌相的改善。本研究初步開發出一種具有改善腸內菌相並有促 進腸道健康潛力的乳酸發酵果汁。中文摘要 .........i
英文摘要 .........ii
目錄 .........iv
表次 .........vii
圖次 .........viii
縮寫對照表 v x
1. 緒論 .........1
1.1 乳酸發酵果蔬汁 .........1
1.2 酵素 .........3
1.3 益生菌 .........3
1.4 乳酸菌 .........4
1.4.1 乳酸菌簡介 .........4
1.4.2 乳酸菌分類.........8
1.4.3 乳酸菌之生理活性.........10
1.5 益生質 .........12
1.6 共生質 .........12
1.7 腸道功能與健康.........13
2. 研究目的 .........15
3. 材料與方法 .........16
3.1 實驗材料 .........16
3.1.1 原料 .........16
3.1.2 試驗菌株 .........16
3.2 試驗設計 .........16
3.3 試驗方法 .........19
3.3.1 建立發酵模式試驗.........19
3.3.1.1 發酵液製作前處理.........19
3.3.1.2 菌株活化.........19
3.3.1.3 pH 值.........20
3.3.1.4 可溶性固形物 .........20
3.3.1.5 可滴定酸度 .........20
3.3.1.6 發酵液酵母菌、醋酸菌及總乳酸菌分析.........21
3.3.1.7 發酵液產率測定 .........22
3.3.1.8 感官品評 .........23
3.3.2 體外試驗 .........23
3.3.2.1 乳酸菌菌株之活化 .........23
3.3.2.2 樣品前處理 .........23
3.3.2.3 吸光值試驗.........24
3.3.3 體內試驗 .........24
3.3.3.1 飼料配製 .........24
3.3.3.2 管餵樣品準備.........25
3.3.3.3 動物飼養.........25
3.3.3.4 糞便取樣 .........27
3.3.3.5 糞便水分含量測定.........27
3.3.3.6 糞便 pH 值測定.........27
3.3.3.7 糞便菌相分析.........28
3.3.4 統計分析 .........29
4. 結果與討論.........30
4.1 合適發酵方法和條件之評估.........30
4.1.1 乳酸發酵及混合式發酵法之比較.........30
4.1.2 不同原料基質發酵對綜合果汁之影響 .........38
4.1.3 原料的比例與種類對發酵成品風味之影響 .........44
4.1.4 添加果膠酶對發酵液之影響.........48
4.1.5 添加異麥芽寡糖對發酵液之影響.........55
4.1.6 發酵果汁配方之評估.........61
4.2 發酵果汁之生理活性評估.........64
4.2.1 體外試驗 .........64
4.2.2 體內試驗 .........66
5. 總結論 .........73
6. 參考文獻.........74
7. 附錄—感官品評問卷 .........8
The Effect of Microwave Treatment on The Esterification in Kaoliang Spirit.
No full textNew distilled kaoliang spirit must undergo aging process in order to reduce the pungent taste after drinking ,remove the off-flavor and make the aroma more attractive to consumers. Esters are the key com-pound of alcohol beverages aroma because their low threshold and distinctive flavor. Not only the esters in alchol beverages increase their content via esterifition ,esters and other compounds also reach equi-librium state during the aging process.
Aging process is a time and space-consuming operation. Reducing the aging time via other treatment means lowering the production cost. Microwave irradiation has been utilized in many organic synthesis reac-tion because it can accelerate the reaction rate in short time by inducing compounds to vibrate. The aim of this master thesis is to shorten the aging process time in order to reduce the production cost. Kaoliang spirit is the sample in this experiment because it is one of the most character-istic alcohol beverages and the concentration changes ofethyl acetate -the main aromatic compound in kaoliang spirit is the indicator of the effect of microwave treatment.
The results of batch-microwave treatment shows that microwave treatment can accelerate the esterification reaction rate about 3.35 times without containing ethyl acetate at the beginning. However, because micro- wave improve the forward and reverse reaction rate simul-tane-ously ,the esterification rate gets slower while the ethyl acetate concen-tration accumulated. It is reasonable to doubt that the way to affect the concentration of ethyl acetate and acetic acid is not mainly via esterifi-cation because of the unequal change between ethyl acetate and acetic acid in the continuous experiment. Therefore, the forward and reverse reaction rate are raised while applying continuous operation system and microwave treatment to new distilled kaoliang spirit ,causing the dif-ferent concentration change between ethyl acetate and acetic acid. But the equilibrium constant of reaction turned down from 4.57 to 3.43 after microwave treatment. It reveals that microwave treatment can urge the reaction to equilibrium.
In conclusion, although that the continuous operation system used in this experiment can help the reaction reach equilibrium quickly but is not an appropriate operation way to increase the ethyl acetate concen-tration. However, the flavor quality of aged Chinese spirit would be a harmonization of many compounds, could not just rely on the concen-tration of a particular compound. Hence, a sensory experiment is needed in order to check the effect of continuous microwave treatment on kao-liang spirit in the future.新製好的酒品需經陳化處理，使新酒原先入口後的刺激感降低，雜味消除讓香味醇正，並使酒香更加誘人。而酯類由於其使人類感受到的閾值濃度低，且又帶有特殊香氣，往往是影響酒香的主要關鍵成分。在陳化過程中，酒液中的酯類能再藉由酯化反應增加含量，並與其他物質達成平衡而使整體風味更加圓熟。
不過陳化步驟耗時且占空間，如果可利用其他處理方式來縮短陳化所需的時間即可降低生產成本。微波因為能使分子快速震動，可在短時內提高反應速率，已利用在促進多種有機合成反應中。本實驗希望藉由微波處理來縮短陳化處理所需要的時間，以達節省酒類生產製作成本。並以亞洲華人地區的特色酒品－高粱酒為樣品，並以高粱酒主要香氣成分的乙酸乙酯含量變化，作為微波加速陳化操作效果之指標。
實驗先以批式操作模擬酒液來探討微波操作對於正向酯化反應的促進效果。由實驗結果可得知，在樣品起始無乙酸乙酯存在下，微波操作可以提升酯化反應速率達3.35倍。但隨乙酸乙酯含量增加，微波同時也促進逆向反應進行，而降低乙酸乙酯生成速率。實驗最後以新酒進行實驗，由連續式實驗中乙酸乙酯與醋酸的濃度改變趨勢不一致可合理懷義，影響酒液中乙酸乙酯濃度變化的因素眾多，應非只由乙醇與醋酸行正逆酯化反應而已。因此以本實驗的循環攪拌系統搭配微波處理，會同時加速多種反應進行，而使乙酸乙酯與醋酸濃度產生不一致的變化。而雖然新酒中乙酸乙酯濃度在連續式微波處理後降低，但以酯化反應平衡常數而言，經微波處理後樣品的反應平衡常數從4.57下降至3.43，較未處理前接近老熟酒的反應平衡常數3.0352。由此可知微波處理可以促進反應到達平衡狀態。
綜合實驗結果：若針對提高酒液內乙酸乙酯含量，則本實驗所採用之連續式微波系統並非是合適的操作方式。但事實上陳化酒的香氣品質優劣，可能需請品酒師品評，感受酒液內芳香物質的整體協調性而決定，並非以單一香氣成分濃度高低而決定。因此，未來可搭配品評試驗來確認連續式微波處理對高粱酒品質之影響。表目錄 III
圖目錄 IV
中文摘要 V
Abstract VI
第一章、前言 1
第二章、文獻回顧 2
一、高粱酒 2
(一)酒精飲料簡介 2
(二)酒精飲料分類 2
(二)酒類呈味影響因素 5
(三)高粱酒製程 9
二、微波 11
(一)微波簡介 11
(二)微波原理 11
(三)微波應用實例 12
第三章、材料與方法 14
一、實驗材料 14
(一)試藥 14
(二)樣品 14
二、實驗器材 14
三、實驗方法 19
1.批式微波酯化反應 19
2.連續式酯化反應 19
2-1溫度效應 19
2-2濃度效應 20
2-3新酒連續式處理 20
四、分析方法 22
(一) Ethyl acetate乙酸乙酯濃度測定 22
(二) Acetic acid醋酸濃度測定： 22
第四章、結果與討論 24
一、模擬酒液模型建立 24
二、確立高粱酒陳化指標 26
三、批式微波操作對於乙酸乙酯生成速率之影響 28
(一)結果與討論 28
(二)小結 30
四、連續式微波處理對於乙酸乙酯生成速率之影響 38
(一) 結果與討論 38
(二) 小結 40
五、連續式微波處理對於新酒中酯類濃度之影響 46
(一) 結果與討論 46
(二) 小結 48
第五章、總結 53
參考文獻 54
一、中文參考書目 54
二、英文參考書目 5
Effect of insulin on the proliferation of colon cancer cells and the underlying mechanism, and the intervention effect of the ginsenoside compound K
No full textThe progression of multiple cancers, including liver, pancreatic, breast, colon/rectum and bladder cancer may exhibit a direct correlation between combination of hyperglycemia and hyperinsulinemia in diabetes mellitus (DM) patients. The survival rate of the patients with DM and colorectal cancer (CRC) is much lower than that of patients with CRC only, and hyperglycemia may be a mediator to induce hyperinsulinemia and increase cancer risk. Compound K (CK) is a metabolite of panaxndiol ginsenosides in the intestinal and possesses anti-cancer properties such as anti-proliferation, cell cycle arrest and pro-apoptotic effect on CRC cancer. CK has been considered to exhibit anti-cancer and improving diabetes. Therefore, the human colon cancer HCT 116 cell line was used to investigate the effect of insulin on cell proliferation and its underlying molecular mechanism, as well as the effect of CK on insulin-induced cell proliferation and metastasis.
The results showed that insulin can promote the cell proliferation, migration and anchorage-independent growth of HCT116 cells. Insulin enhanced the expression of mRNA and activity of matrix metalloproteinases-2 (MMP-2) and MMP-9, leading to a rise of the metastatic ability in HCT 116 cells. The results also showed that insulin combined with its insulin receptor (IR) to activate insulin receptor substrate 1 (IRS-1), which altered its downstream signals of PI3K-Akt and MAPKs to cause a rise of cell proliferation-associated transcription factor, HSP27. To further verify the effects of the major signals on insulin-promoted cell proliferation, the protein expressions of PI3K and MAPKs were inhibited. It was found that the suppressions of PI3K and ERK signals can attenuate insulin-induced proliferation. Furthermore, insulin activated by PI3K-Akt pathway caused an increase of cell proliferation-related protein expression (e.g. phosphorylation of GSK3β and mTOR) to increase the level of cell cycle-related protein cyclin D1 and to decrease c-Myc expression. However, insulin also stimulated the expression of p53 protein, suggesting that p53 has a feedback regulation to stimulate a dramatic increase, leading to inhibition of cell proliferation. The results indicated that CK can significantly reduce the migration ability of the insulin-induced HCT 116 cells, and it slightly reduced the effect of cell proliferation and anchorage-independent growth.
In conclusion, insulin can activate the downstream molecules through IRS-1 signal and increase the expression of PI3K-Akt and MAPKs, which eventually result in the effects of cell proliferation and cell cycle-related protein and enhance cell proliferation and metastasis of CRC. Therefore, it is suggested that ginsenoside CK has the potential to reduce the effects of insulin-induced cancer progression.糖尿病患者因體內合併高血糖及高胰島素血症之症狀，可能與多種癌症 (肝癌、胰臟癌、乳癌、大腸癌及膽囊癌等) 之進展有直接之關聯性。研究指出，罹患糖尿病之大腸癌患者存活率，遠低於未罹患糖尿病之大腸癌病患，而高血糖可能為高胰島素血症等誘發因子之媒介，因而增加癌細胞增生及侵入轉移能力。Compound K (CK) 為二醇型人參皂苷於腸道中之降解產物。文獻指出，CK 可抑制大腸癌之細胞增生及細胞週期進展且促細胞凋亡。許多研究指出，人參皂苷CK 具抗癌及改善糖尿病之功效。因此本研究分為兩部分進行探討：(一) 胰島素對大腸癌細胞增生之影響及其機轉探討；(二) CompoundK 抑制胰島素誘導大腸癌細胞增生及侵入轉移之影響。
研究結果顯示，胰島素會加速 HCT 116 大腸癌細胞之增生、移行及非依附性生長 (anchorage-independent growth) 能力 同時促基質，金屬蛋白 2/9 (matrix metalloproteinase-2/9, MMP-2/9) 之 mRNA 及活性表現，而使癌細胞之侵入轉移能力上升。深入了解相關分子機制後發現胰島素可與其專一性細胞接受器 (insulin receptor, IR) 結合，因而活化胰島素接受器受質蛋白 1 (insulin receptor substrate 1, IRS-1)而影響其下游途徑 PI3K-Akt 及 MAPKs 等相關訊息傳遞路徑，導致與細胞增生相關之轉錄因子 HSP27 之轉錄活性上升。為了解其中影響胰島素促進癌細胞增生之主要訊息因子，實驗進一步抑制 PI3K及 MAPKs 等相關訊息蛋白之表現。結果發現，抑制 PI3K 及 ERK能顯著減弱胰島素促癌之細胞增生作用。另外，本研究亦發現，胰島素可能透過活化 PI3K-Akt 訊息傳遞路徑導致細胞增生相關之蛋白GSK3β 及 mTOR 磷酸化表現上升，並促使與細胞週期相關之蛋白cyclin D1 表現上升及 c-Myc 表現量下降；然而，胰島素亦會提升抑癌蛋白 p53 之表現量，推測此現象為 p53 蛋白本身回饋調控機制促使其表現大量增加並反制細胞大量增生之結果。人參皂苷相關功效評估指出，CK 能顯著降低胰島素誘導之大腸癌細胞爬行能力，而對於細胞增生及非依附性生長則可些微降緩。
綜合上述，胰島素可透過 HCT 116 細胞其專一接受器活化下游相關訊息蛋白，並可活化 PI3K-Akt 及 MAPKs 及增加其表現量，進而影響與細胞增生及週期相關之訊息傳遞，促進大腸癌細胞增生並且影響其侵入轉移能力。而人參皂苷 CK 可能具有減緩胰島素促癌現象之潛力。全文摘要.............i
Abstract .............iii
目次.............v
圖次v ix
附圖.............xi
表次.............xii
縮寫表.............xiii
前言.............1
第一章：文獻回顧 .............3
壹、 糖尿病 .............4
一、 背景 .............4
二、 定義 .............4
貳、 糖尿病與癌症之相關性.............7
一、 高糖與癌症之相關性.............10
二、 胰島素與癌症之相關性.............12
參、 大腸癌 .............18
一、 癌症 .............18
二、 大腸癌 .............18
三、 大腸癌之分期階段.............21
四、 大腸癌之流行病學.............21
肆、 高血糖及高胰島素血症與大腸癌之關係 .............23
伍、 癌症之細胞週期及增生.............26
一、 細胞增生.............26
二、 細胞週期 .............26
陸、 癌症之侵入轉移.............31
一、 轉移之前提條件 .............31
二、 轉移之起始與擴散 .............31
三、 轉移性定殖 (Metastatic colonization) .............32
捌、 樣品介紹.............34
一、 人參之簡介 .............34
二、 人參皂苷 - Compound K .............35
捌、 研究目的 .............38
玖、 研究架構.............40
第二章、胰島素對大腸癌細胞增生之影響及其機轉探討 .............41
摘要 .............42
Abstract .............43
前言.............45
實驗架構 .............47
材料與方法 .............48
結果.............55
一、 胰島素對人類大腸癌 HCT 116 細胞存活率之影響 ..........55
二、 胰島素對 HCT 116 細胞增生之影響.............55
三、 胰島素對 HCT 116 細胞長期生長之影響 (7 天) .............55
四、 胰島素對 HCT 116 細胞中 IR 途徑相關蛋白表現之影響...56
五、 胰島素對 HCT 116 細胞中 PI3K-Akt 途徑下游相關蛋白表現之影響 ..... 57
六、 胰島素對 HCT 116 細胞中 MAPKs 途徑相關蛋白表現之影響 ........57
七、 胰島素對 HCT 116 細胞中細胞週期相關之 c-Myc 及cyclin D1 蛋白表現之影響...... 57
八、 胰島素對 HCT 116 細胞中 c-Myc 及 HSP27 轉錄因子轉位活性之影響 ...... 58
九、 胰島素促進 HCT 116 細胞增生之主要分子機轉分析 ......58
討論 .............60
第三章、Compound K 抑制胰島素誘導大腸癌細胞增生及侵入轉移之影響.............79
摘要 .............80
Abstract .............81
前言 .............82
實驗架構 .............83
材料與方法.............84
結果 .............93
一、 胰島素對 HCT 116 細胞增生之影響 .............93
二、 胰島素對 HCT 116 細胞移行能力之影響.............93
三、 胰島素對 HCT 116 細胞基質金屬蛋白酶 2 及 9 mRNA表現之影響.......93
四、 胰島素對 HCT 116 細胞基質金屬蛋白酶 2 及 9 活性之影響..........94
五、 Compound K 對 HCT 116 細胞存活率之影響 .............94
六、 Compound K 對胰島素誘導 HCT 116 細胞增生之影響 ..94
七、 Compound K 對胰島素誘導 HCT 116 細胞移行之影響 ..95
八、 Compound K 對胰島素誘導 HCT 116 細胞基質金屬蛋白酶 2 與 9 活性之影響 ............95
討論 .............97
總結論.............111
參考文獻.............11