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建立一套新穎轉基因細胞懸浮培養系統生產嵌合竹嵌紋病毒顆粒
No full text隨著基因工程工具與高通量篩選技術時代的來臨，帶來分子生物與生物醫學領域的重大革新，次單位疫苗的發展更取代傳統動物與細胞培養所生產的疫苗，過去20年來由植物衍生的疫苗由於便宜與方便管理是一套迷人的策略; 主要區分為穩定或暫時表現的兩套系統不同的系統各有優點與其限制轉基因植物屬於穩定表現的系統，可以持續且穩定的生產疫苗，但表現量相對暫時表現系統來的略低; 暫時性表現系統，透過融合胜肽於病毒外鞘蛋白，所形成的嵌合型病毒顆 (chimeric virus particles, CVPs) 可以迅速生產出高產量的疫苗，但由於病毒的基因體沒有插入植物基因體，因此相較於轉基因植物無法持續且穩定的生產。本研究建立一套新穎的植物懸浮細胞培養系統來生產疫苗，以竹嵌紋病毒外鞘蛋白N端融合一段來自口蹄疫病毒的病毒蛋白1 (VP1) 之抗原決定基，將此嵌合竹嵌紋病 (chimeric BaMV-CP) 與野生型竹嵌紋病毒株 (wild-type BaMV) 之cDNA轉殖Nicotiana benthamiana，利用轉殖株葉片建立懸浮培養的轉基因菸草細胞，利用西方墨點法與酵素結合免疫吸附分析 chimeric BaMV-CP 與 wild-type BaMV 的表現量，結果顯示轉基因懸浮培養細胞株 BdT38 與 BdT19每20克懸浮細胞可以分別得到1.5 與2.1 mg 的嵌合竹嵌紋病毒顆粒 (CVPs), 免疫電子顯微鏡中VP1 抗原決定基存在於嵌合竹嵌紋病毒顆粒的表面上，施打嵌合竹嵌紋病毒顆粒於天竺鼠可有效產生中合性抗體。為了簡化純化嵌合竹嵌紋病毒顆粒，我們發展一套利用分子篩層析取代傳統離心的純化病毒的原型流程，結果顯示此方式可有效從菸草懸浮細胞回收到嵌合竹嵌紋病毒。我們研究考量經濟效益，而發展出一套以植物作為生產免疫胜肽 (immunopeptide) 疫苗的新平台，並嘗試簡化純化病毒的步驟，以增加疫苗大規模生產的效率。Advent of genetic engineering tools and high-throughput screening technologies have revolutionized in the field of molecular biology and biomedical science. As a consequence, these technologies enabled the development of subunit vaccines to replace traditional animal and bacterial cell-cultures based vaccines. Similarly, last two decades plant derived antigens have been a fascinating strategy to use as vaccines candidates due to low cost and convenient administrations. Production of vaccine antigens in plants is often achieved through stable or transient expression systems. However, these technologies have certain advantages and limitations; transgenic plants can produce vaccine antigens consistently but yield is very low, in case of transient expression, in which the fused peptide expresses on the chimeric virus particles (CVPs) combined with rapid and high yield production in whole-plants system but not consistent due to viral genome is not integrated into the plant genome.
In this study, a novel vaccine production method is developed using plant cell suspension culture system. We utilized this unique transgenic cell-suspension cultures derived from transgenic Nicotiana benthamiana leaves expressing wild-type or chimeric Bamboo mosaic virus (BaMV) transgene carry viral protein 1 (VP1) epitope of foot and mouth disease virus (FMDV) at the N-terminus of coat protein (BVP1). The expression of BVP1 protein was detected in cell-suspension cultures by Western blot and ELISA. Subsequently, a high level of chimeric BaMV particles (CVPs) were purified from BdT38 and BdT19 transgenic cell-suspension cultures co-expressing silencing suppressor protein P38 and P19, respectively (~ 1.5 or 2.1 mg/20 g of suspended fresh weight biomass) when compared with transgenic line BVP1-16-7, in which this silencing suppressor protein expression is absent. Furthermore, immunoelectron microscope analysis revealed the VP1 epitope expression on the surface of CVPs. Subsequently, guinea pigs vaccinated with these CVPs particles have generated neutralizing antibodies.
We have developed a prototype protocol to simplify the purification procedure using size-exclusion chromatography to replace the traditional centrifugation based virus purification. Our results showed a remarkable recovery of CVPs from cell-suspension cultures. VP1 epitopes were displayed on the surface of CVPs. Our studies provide insight to generate immunopeptide vaccines in a cost effective manner, as well as streamline the methods for a large-scale production using natural plant-based milieu that offers an alternative and efficient vaccines source.Contents
I. Abstract
Chinese....................................................i
English...................................................ii
II. Background ...........................................................1
Cell-suspension culture....................................3
Therapeutic recombinant protein expression.................4
Vaccine antigens in cell-suspension culture................5
Silencing suppressor proteins..............................6
Foot and mouth disease virus...............................7
Bamboo mosaic virus........................................7
III. Research topics.......................................9
Chapter I
Production of chimeric Bamboo mosaic virus particles in the transgenic
Nicotiana benthamiana plant cell culture…..................9
1. Abstract ..............................................10
2. Introduction ..........................................11
3. Materials and Methods..................................14
3.1 Plant material and callus induction...................14
3.2 Cell-suspension culture...............................14
3.3 Determination of growth curve of suspended biomasses..15
3.4 Western blot analysis.................................15
3.8 ELIS assay............................................16
3.9 Purification chimeric Bamboo mosaic virus ............16
4.Results.................................................18
4.1 Construction of chimeric BaMV expression cassettes....18
4.2 Establishment of cell-suspension culture..............18
4.3 Expression of chimeric BaMV coat protein in cell-suspension culture........................................18
4.4 Effect of light on BaMV expression in cell-suspension cultures..................................................19
4.5 Purification of CVPs..................................20
5. Discussion............................................21
6. Table..................................................23
7.Figures.................................................24
Chapter II
A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles........30
1. Abstract ..............................................31
2.Introduction............................................32
3.Materials and Methods...................................34
3.1 Construction of recombinant expression cassettes......34
3.2 Plant material and callus induction...................34
3.3 Plant cell-suspension culture.........................34
3.4 Growth determination .................................35
3.5 Immunoblotting assay..................................35
3.6 Northern blot analysis................................36
3.7 Small RNA detection by Northern blot..................36
3.8 Stability of chimeric genomic BaMV RNA analysis by RT-PCR.......................................................37
3.9 ELISA.................................................37
3.10 Purification of BaMV CVP.............................38
3.11 Transmission electron microscopy of immunogold-labelled complexes.................................................39
3.12 Vaccination of guinea pigs...........................39
3.13 ELISA titration of CVP-immunized guinea pig sera.....39
3.14 Serum neutralization assay...........................40
3.15 Statistical analysis.................................40
4.Results.................................................41
4.1 Construction of chimeric BaMV expression cassettes....41
4.2 Establishment of transgenic cell-suspension culture...41
4.3 Transgenic cell-suspension cultures expressing chimeric
BaMV coat protein.........................................42
4.4 Detection of silencing suppressor protein.............42
4.5 Quantification of BVP1................................43
4.6 Detection of chimeric BaMV genomic RNA................43
4.7 Detection of small RNA................................44
4.8 Stability of chimeric BaMV genome in transgenic cell-lines.....................................................45
4.9 Characterization of CVPs purified from suspended biomass...................................................45
4.10 Elicitation of specific antibodies in BVP1-immunized guinea pigs...............................................46
4.11 Serum neutralization assay...........................47
5. Discussion ............................................48
Table 1...................................................54
Table 2...................................................55
Figures...................................................56
Chapter III
Purification of chimeric Bamboo mosaic virus from transgenic cell-suspension cultures by size-exclusion chromatography............................................65
1. Abstract...............................................66
2. Introduction...........................................67
3. Materials and Methods..................................70
3.1 Plant materials for viral particles purification......70
3.2 Establishment of callus and cell-suspension culture...70
3.3 Precipitation of wild type and chimeric BaMV..........71
3.4 Gel filtration........................................71
3.5 SDS-PAGE and Immunoblotting analysis..................71
3.6 Immunogold labelling and Electron microscopy..........72
4. Results................................................73
4.1 Standardization of chromatography conditions for
the purification of BaMV-S................................73
4.2 Chimeric Bamboo mosaic virus purification.............73
4.3 Western blotting analysis.............................74
4.4 Characterization of eluted chimeric BaMV particles....74
5. Discussion.............................................76
Figures...................................................79
IV. References............................................8
芝麻半胱氨酸蛋白酶抑制劑特性研究及腐霉菌二十碳五烯酸合成路徑中相關基因之功能鑒定
No full text本論文主要分為兩個主題:
主題一之內容為芝麻半胱氨酸蛋白酶抑制劑之特性研究。植物之半胱氨酸蛋白酶抑制劑是半胱氨酸蛋白酶之抑制劑。根據分子量大小，植物半胱胺酸蛋白酶抑制劑可分為三個群組。芝麻所表達之半胱胺酸蛋白酶抑制劑，其結構上顯示由N端與C端二個domain所組成，屬於植物半胱胺酸蛋白酶抑制劑家族第二群分子，然而至今仍然未能確定C端之功能。為了探討此二個domain的功能，將此二個domain分別以大腸桿菌表現重組蛋白，並探討其生理作用與可能之應用。
主題二之內容則是探討腐霉菌二十碳五烯酸合成路徑中相關基因之功能鑒定。在此部分的研究中，發現了五個基因參與腐霉菌二十碳五烯酸之生合成。而PaELO2與PaELO3具有較高之碳鏈延長活性，顯示可利用基因操作之方式將之運用於酵母和植物的代謝工程，生產長鏈多元不飽和脂肪酸以供保健食品之應用。The thesis is divided into two parts.
Part I of this thesis discusses the characterization of group II phytocysatin from sesame. Phytocystatins, a group of proteins from plants specifically inhibited cysteine proteinase. According to the molecular weight, phytocystatins are classified into three distinct groups. Sesame cystatin containing an additional C-terminal (Ct) extension belongs to group II phytocysatin. However, the Ct extension with unknown role is found in sesame cystatin. To gain insights into the functions of the two domains in SiCYS, these recombinants were explored the physiological roles and potential applications.
Part II of this thesis deals with the genes involved the biosynthesis of eicosapentaenoic acid from Pythium aphanidermatum. In the present study, five novel genes were cloned from the EPA rich microbe P. aphanidermatum. The functional analysis indicated those genes involved a complete pathway for EPA biosynthesis. The PaELO2 and PaELO3 possess higher elongated activity. It could be used for metabolic engineering of yeast and plant to produce VLCPUFAs for nutraceutical applications.Abstract I
List of Figures III
List of Tables V
Table of Abbreviations VI
Part I
Sesame group II phytocystatin characterization studies
Chapter 1: Introduction 1
Chapter 2: Materials and Methods 4
Chapter 3: Results 8
Chapter 4: Discussion and conclusions 12
Reference 16
Part II
Identification and characterization of genes from Pythium aphanidermatum involved in eicosapentaenoic acid biosynthesis
Chapter 1: Overview 28
Chapter 2: Literature Review
2.1 Fatty Acid Classification and Nomenclature 30
2.2 Health benefits of long-chain polyunsaturated fatty acids (LC-PUFAs) 33
2.3 Fatty acid biosynthesis 35
2.4 Fatty acid desaturase 38
2.5 Fatty acid elongase 40
2.6 Aims of this work 41
Reference 42
Chapter 3: Identification and characterization of desaturases from Pythium aphanideratum involved in eicosapentaenoic acid biosynthesis
3.1 Introduction 51
3.2 Materials and Methods 53
3.3 Results 56
3.4 Discussion 59
Reference 63
Chapter 4: Identification and characterization of elongases from Pythium aphanideratum involved in eicosapentaenoic acid biosynthesis
4.1 Introduction 81
4.2 Materials and Methods 82
4.3 Results 83
4.4 Discussion 85
Reference 89
Chapter 5: Conclusions 10
菸草異檸檬酸脫氫酶對於竹嵌紋病毒複製的影響
No full text病毒通常藉由宿主細胞中的組成來完成其感染與複製行為，因此近年來許多研究致力於病毒與其宿主因子關係之探討。竹嵌紋病毒( (Bamboo mosaic virus)) 為Potexvirus 屬，是長絲狀單鏈正意股RNA病毒，基因體全長約 6.4-kb。本實驗室先前研究利用農桿菌轉染於菸草中表現竹嵌紋病毒複製酵素，經由免疫沉澱法及串聯質譜儀找到多個可能與竹嵌紋病毒複製酵素產生交互作用之菸草宿主因子，如：AtClpC、pleiotropic drug resistance like protein、Scarecrow-like protein 5、isocitrate dehydrogenase ( (ICDH)) 、Calcium-transporting ATPase 4，endoplasmic reticulum-type-like。利用病毒誘導靜默法( (virus-induced gene silencesilencing, VIGS)) 及西方墨點法( (Western blot)) ，發現ICDH蛋白質表現量調降後，病毒的殼蛋白質增加。而在菸草原生質體實驗中，過量表現ICDH時，病毒殼蛋白質的累積量則明顯受到抑制，顯示ICDH於植物中可能具有抗病毒之功能。利用點突變的方式，分別改變ICDH的催化活化位NbICDH-Y205A序列，使其失去蛋白質催化活性，利用菸草原生質體實驗，過量表現突變蛋白質，相較於野生型NbICDH，對於竹嵌紋病毒複製影響並無差異，可見NbICDH對於竹嵌紋病毒複製的調控與其蛋白質的酵素活性無關。有關NbICDH抑制病毒累積的機轉有待進一步的釐清。Viruses recruit many of the host cell factors to complete infection cycle. Recently, many studies have committed to explore the relationship between the virus and its host factors. Bamboo mosaic virus ( (BaMV)) , belonging to the genus Potexvirus, is a single-stranded 6.4-kb positive sense RNA virus.. In our lab, BaMV relpicase complex was prepared from Nicotiana benthamiana that had been Agroinfiltrated agroinfiltrated with BaMV replicase-expression cassette, followed by partial purification using immunoprecipitation. Several speculative host factors were subsequently identified by LC-MS/MS, including AtClpC, pleiotropic drug resistance like protein, scarecrow-like protein 5, isocitrate dehydrogenase ( (NbICDH)) and Calcium-transporting ATPase 4. It was found that when NbICDH was down-regulated by virus-induced gene silence silencing ( (VIGS)) , the accumulation of BaMV coat protein was increased. NbICDH Overexpression overexpression NbICDH in protoplasts, decreased the accumulation of BaMV coat protein was decreased. The result suggests that NbICDH may have antiviral function in N. benthamiana. The catalytic site residue Y205 of NbICDH were was replaced by alanine by mutagenesis to examine the involvement of the enzyme's catalytic activity in BaMV replication. Overexpression of the mutant proteins in protoplasts showed the same inhibition effect on the accumulation of BaMV coat protein as the wild-type NbICDH. This result suggests that the catalytic activity of NbICDH is not required for decreasing the viral replication. In the future, the regulation of BaMV replication by NbICDH should be demonstrated.目次
國立中興大學生物科技學研究所 i
摘要 i
Abstract ii
目次 iii
壹、前言 1
一、竹嵌紋病毒 1
二、宿主因子 1
三、研究背景介紹 3
四、本實驗之目的 3
貳、材料與方法 4
一、材料 4
( (一)) 溶液 4
( (二)) 培養基 6
( (三)) 菌株與質體 6
( (四)) 引子 6
二、方法 7
( (一)) pTRV2-NbICDH 之構築 7
( (二)) 農桿菌接種 7
( (三)) 病毒接種 8
( (四)) 竹嵌紋病毒病毒累積量之分析 9
﹝a﹞植物總量RNA萃取 9
﹝b﹞反轉錄作用 ( (Reverse transcription)) 10
﹝c﹞聚合酶鏈鎖反應( (PCR)) 10
﹝d﹞即時聚合酶鏈鎖反應 ( (Real-time PCR)) 11
﹝e﹞病毒鞘蛋白質含量分析 11
( (五)) 過量表現NbICDH 12
( (a)) NbICDH全長及pBI221-NbICDH之構築 12
( ( c )) 鞘蛋白質濃度分析 14
( (六)) 點突變 14
( ( a )) 點突變構築 14
( ( b )) 原生質體分析 14
參、結果 15
一、菸草異檸檬酸脫氫酶基因靜默對於竹嵌紋病毒鞘蛋白質累積量之分析 15
( (一)) 病毒誘導基因靜默VIGS 15
( (二)) 聚合酶鏈鎖反應與即時反轉錄後聚合酶鏈鎖反應分析NbICDH基因靜默現象 1516
( (三)) 竹嵌紋病毒鞘蛋白質累積量分析 1617
二、菸草異檸檬酸脫氫酶基因過量表現對於竹嵌紋病毒鞘蛋白質累積量之分析 17
( (一)) 在菸草原生質體中過量表現pNbICDH對竹嵌紋病毒複製之影響 17
三、病毒誘導靜默菸草NbICDH對於FoMV鞘蛋白質累積量影響 18
四、點突變 18
肆、討論 20
一、竹嵌紋病毒宿主因子篩選 20
二、異檸檬酸脫氫酶 20
伍、圖表 23
陸、參考文獻 44
一、中文參考文獻 44
二、英文參考文獻 4
混合農藥對微生物毒效之影響研究
No full textAgriculture is an important industry for people, with advances in technology and medicine, the ways of agricultural production have gradually changed. People use pesticides to avoid pests and diseases from crops. However, the excessive use of pesticides let people began thinking whether pesticides cause negative impact on people and environment, especially the toxicity of mixtures, it's the focus of many studies.
Carbendazim, procymidone and triforine are fungicide that commonly used on agricultural. Caffeic acid and cinnamic acid are commonly found soil rhizosphere phenolic compounds. This five pesticides above are common found in our life, but we know little about their single and mixture toxicity. So in this study, we discuss carbend- azim, procymidone, triforine, caffeic acid and cinnamic acid single and mixture toxicity by botrytis cinerea and Micrtox antimicrobial test. Further use UV spectrophotometer to compare single and mixture absorption spectra. Trying to find out if structure change after pesticides mix. Attempt to identify and determine the parameters of the experimental toxicity data then predict the toxicity of linear equations. Finally, using Autodock Tools to simulate bacteria and pesticides relationship between combine position, toxicity and binding energy. Results show that carbendazim mix with triforine will produce antagonism leads to reduced toxicity.農業生產一直是人類賴以生存的產業，隨著科技及醫學的進步，人們對於農業生產的方式也逐漸改變。人們使用農藥來確保作物不受病蟲害影響而降低產量。然而農藥過度使用的結果，卻也讓人們開始反思農藥對人們及環境是否會帶來負面的影響，尤其是農藥混合之後的毒性，更是目前許多研究的重點。貝芬替、撲滅寧、賽福寧是農業上常用的殺菌劑，而咖啡酸及肉桂酸則是根圈土壤中常見的酚酸化合物。以上五種農藥皆是日常生活中常見的常接觸的化合物，然而我們對其單一或混合毒性的了解卻所知甚少。因此在本研究中，分別利用灰黴菌及 Microtox 發光菌之抑菌實驗討論了貝芬替、撲滅寧、賽福寧、咖啡酸及肉桂酸單一及混合的毒性。使用紫外線可見光光譜儀觀察單一及混合的吸收光譜的差異，希望可以得到農藥混合之後是否會有結構上的改變。嘗試藉由定量構效關係(Quantitativestructure-activity relantionship , QSAR)電子參數的角度，能夠找出決定毒性的參數與實驗值毒性數據線性關係之毒性預測方程式。最後使用Autodock Tools 嘗試模擬發光菌與農藥結合的情況，期望可以得知農藥在發光菌上結合位置和能量與毒性的關係。從結果中可以得知貝芬替混合賽福寧後會產生拮抗作用導致毒性降低。第一章 緒論........1
1-1 研究動機........1
1-2 研究目的........3
第二章 理論背景........4
2-1 評估有機化合物混合毒性之模式與種類........4
2-2 密度泛函理論........9
第三章 實驗與計 算方法........11
3-1 殺菌劑製備 及抑菌實驗........11
3-1-1 殺菌劑製備........11
3-1-2 抑菌實驗........11
3-2 Microtox(急毒性分析儀)實驗........12
3-2-1 Microtox Model 500........12
3-2-2 樣品溶液配製........13
3-2-3 Microtox Model 500 實驗方法........13
3-3 紫外線可見光光譜儀實驗........14
3-3-1 紫外線-可見光光譜法（UV-Visible Spectrophotometer）........14
3-3-2 操作方法與設定........15
3-4 分子參數計算........15
3-4-1 結構優化計算........15
3-4-2 得失電子計算........16
3-4-3 參數計算........17
3-4-4QSAR 分析........17
3-5 AutoDock 計算........18
第四章 結果與討論........20
4-1抑菌實驗........20
4-1-1 灰黴菌抑菌實驗經 48 小時之後結果........21
4-1-2 灰黴菌抑菌實驗經 72 小時之後結果........32
4-2 Microtox(急毒性分析儀)實驗........46
4-3 紫外光分光光度計實驗........51
4-4 以密度泛函理論為基礎的定量結構活性關係........69
4-5AutoDock 結合能與結合位置........104
第五章 結論........110
參考文獻........11
Regulatory effect of HMGB1 on the process of epithelial-mesenchymal transitionand reversal potential role of glycyrrhizin on E-cadherin in prostate cancer cells
No full textProstate cancer is the first cause of male cancer incidence in the developed countries and is hard to eradicate because of the high metastasis and easy relapse. Therefore, the prevention and therapy of prostate cancer is an important study. Tumor with rapid growth is one of the characteristics in cancer metastasis, resulting from growing environments of hypoxia, starvation and chronic inflammation within the tumor tissue. The stress stimulation during harsh environment not only causes to get nutrients and growing space but also to invade surrounding tissue into the circulatory system. Epithelial-mesenchymal transition (EMT) is the first step in the process of cancer cells with invasion and tumor metastasis, which can help cell adhesion decrease the ability of cell-cell junction and generate cell polarity. The effects result in tumor escape form tumor tissue and migrate, eventually leading to invasion and metastasis. HMGB1plays a role of non-histone chromatin binding nuclear protein and a damage-associated molecular pattern (DAMP), which is released to extracellular by active and passive modes during cell damage. Thus, cancer cells can resist environmental stimuli and injury. The aim of this study was to investigate whether HMGB1 expression can affect its release and regulatory ability of EMT to cause cell invasion. In addition, we also explored the reversal potential effect of phytochemical glycyrrhizin (GR) on E-cadherin of prostate cancer cells was also explored.
The results indicated that knockdown of HMGB1 reduced HMGB1 release in prostate cancer cells, leading to inhibition of phosphorylation of Rac1/Cdc42 to promote GSK-3β activity. The level of GSK-3β activation induced snail degradation and reduced its translocation which increased E-cadherin expression and promoted cell adhesion to down-regulate cell polarity and reduce tumor metastasis. In addition to directly regulate E-cadherin expression, HMGB1 was inhibited to indirectly regulate EMT before being reduced EMT relative inflammatory factor release such as IL-6 and IL-8 to avoid a better condition beneficial for tumor metastasis. To further investigated whether HMGB1-induced Rac1/cdc42 signaling is a major regulatory pathway regarding modulation of EMT ability in prostate cancer cells. The serum-free medium was applied to simulate a starvation environment in tumor growth. The results showed that low expression of HMGB1 in prostate cancer cells exhibited poor ability of tumor migration and low expression of EMT-related inflammatory factors. Based on these finding, HMGB1 is a critical EMT regulator. GR is one of the sweet substances in Liquorice. The recent reports showed that GR is able to bind with HMGB1 to inhibit its cytokine activity. We also found that GR suppressed tumor migration and recovered E-cadherin expression in prostate cancer cells.
In conclusion, HMGB1 could be a hallmark to tumor metastasis. Reduction of prostate cancer metastasis occurred through suppression of HMGB1 releasing and recovering. GR is a phytochemical and has capability to bind HMGB1 and to inhibit tumor metastasis. Therefore, it is suggested that GR may have potential of adjuvant chemotherapy and as an adjuvant therapy.攝護腺癌於已開發國家男性癌症罹患率位居首位，具有高度轉移性且易復發，導致難以根治，因此攝護腺癌之預防及治療為相當重要之研究。癌細胞轉移主因來自於快速生長之特性，導致腫瘤組織內部呈現缺氧、飢餓及慢性發炎之生長環境，癌細胞由於惡劣環境壓力刺激而導致其為了獲得更多養分及生長空間，侵入到周遭組織並進入循環系統。上皮間質轉化現象 (epithelial-mesenchymal transition, EMT)之過程為癌細胞具有侵入轉移之首要步驟，此步驟幫助癌細胞減弱細胞與細胞間黏附能力，並產生細胞極性，使癌細胞脫離腫瘤組織並具有移行能力，進而促進侵入轉移進行。HMGB1 (High mobility groupbox-1) 為一種核蛋白，正常情況下扮演非組蛋白染色質結合蛋白(non-histone chromatin binding protein)之角色，然而細胞受到傷害或刺激之情況下會透過主動及被動兩種方式釋放至細胞外，作為損傷相關分子 (damage-associated molecular pattern, DAMP)，幫助細胞抵抗外來環境之刺激及傷害。本研究目的為探討 HMGB1 於 DU 145 攝護腺癌細胞表現之差異是否會影響其釋放及調控其 EMT 能力 進而調，控癌細胞侵入轉移之特性，並探討天然植物化合物甘草甜素(Glycyrrhizin, GR)回復癌細胞 E-cadherin 表現之潛力。
本研究結果指出，利用基因弱化 (Knockdown) 方式降低攝護腺癌細胞中 HMGB1 (High-mobility group box-1)表現量確實顯著減弱HMGB1 之釋放，此結果導致癌細胞之 Rac1/Cdc42 磷酸化表現減弱，而促進癌細胞 GSK-3β 活性，導致 snail 之降解並減少其轉位，而促使 E-cadherin 表現量顯著提升，進而增加癌細胞間黏附能力，導致癌細胞移行能力減弱而達到抑制侵入轉移之能力。Rac1/Cdc42 之活化除了降低 E-cadherin 外，也可降低癌細胞極性 (cell polarity) 之形成，而使細胞無法脫離腫瘤組織進行移動。除了直接調控訊息傳遞路經促使癌細胞 EMT 能力上升外，抑制 HMGB1 也可減少 EMT 相關發炎因子 IL-6 及 IL-8 之表現量，而避免癌細胞處於有利於侵入轉移之微環境。
此外，以無血清環境方式模擬攝護腺癌細胞處於飢餓環境，進一步探討是否於惡劣生長環境下，HMGB1 所誘導之 Rac1/Cdc42 路徑之活化仍是調控癌細胞 EMT 能力之主要訊息傳遞路徑 結果顯示，。在無血清環境下，HMGB1 釋放確實具有活化前述訊息傳遞路徑之能力，並且經由基因弱化而導致 HMGB1 表現量較低之攝護腺癌細胞株 在無血清環境下之移行能力顯著降低 由此數據顯示 HMGB1 在，，癌細胞 EMT 能力之調控扮演重要之角色 甘草中之主要甜味物質為。甘草甜素，近期文獻指出甘草甜素具有補捉 HMGB1 而抑制其細胞激素功能之用途。本研究發現介入甘草甜素能有效抑制攝護腺癌細胞移 行 之 能 力 ， 並 回 復 正 常 HMGB1 表 現 量 之 攝 護 腺 癌 細 胞E-cadherin 表現量。
綜合上述結果 HMGB1 確實可作為癌症侵入轉移能力之指標，，抑制 HMGB1 之釋放或捕捉已釋放 HMGB1 具有抑制攝護腺癌細胞侵入轉移之功能性。天然植物化合物中，甘草甜素可透過螯合HMGB1 而回復攝護腺癌 E-cadherin 表現量，進而降低癌細胞之侵入轉移能力，因此推測甘草甜素具有輔助治療及作為化療藥物佐劑之潛力。中文摘要..........i
英文摘要..........iii
圖次..........ix
附圖..........xii
縮寫表.......... xiii
前言..........1
壹、文獻整理 ..........3
一、攝護腺癌 (prostate cancer) 簡介 ..........4
二、癌症(cancer)與侵入轉移(invasion and metastasis)之簡介 ..........4
三、Epithelail-mesenchymal transition (EMT) 簡介..........5
1. EMT 與侵入 (invasion) 及轉移 (metastasis) 之關係..........5
2. E-cadherin 降解 ..........6
3. EMT 與細胞極性 (cell polarity) ..........6
4. EMT 調控之訊息傳遞 ..........7
四、High-mobility group protein B1(HMGB1)簡介..........21
1. HMGB1 功能 ..........21
2. The receptor for advanced glycation end products (RAGE) ......22
3. RAGE 與 HMGB1 ..........23
4. HMGB1 與癌症..........24
5. 攝護腺癌之 HMGB1 與 RAGE 之臨床研究..........27
6. 以 HMGB1 作為標的於癌症治療之應用..........28
五、甘草..........29
貳、研究目的 ..........37
HMGB1 對於攝護腺癌細胞上皮間質轉化過程之調控及甘草甜素回復 E-cadherin 之潛力 ..........39
?、研究架構 ..........40
肆、材料與方法 ..........41
一、實驗材料 ..........41
1. 細胞株 ..........41
2. 藥品 ..........41
二、實驗方法..........42
1. 細胞培養..........42
2. 細胞繼代與冷凍保存 (貼附型) ..........43
3. 細胞存活率測定 (MTSassay) ..........43
4. 傷口癒合試驗 (wound healing) ..........43
5. 細胞移行試驗 (Transwell assay) ..........44
6. 細胞內 RNA 抽取..........44
7. 合成 cDNA ..........45
8. Polymerase Chain Reaction (PCR) 反應..........45
9. 瓊脂膠體電泳 ..........46
10. 蛋白質之萃取 (貼附型細胞) ..........46
11. 細胞核/細胞質蛋白質之萃取 (貼附型細胞) ..........46
12. 培養基中 HMGB1 蛋白質濃縮 ..........47
13. 聚丙烯醯胺膠體電泳法 (SDS-PAGE) 分析..........47
14. 西方轉漬法 (Western blot assay..........48
15. 螢光免疫染色法 (Immunofluorescencev48
16. 酵素連結免疫吸附測定分析(Enzyme-linked immunosorbent assay, ELISA) ..........49
17. 胞內鈣離子濃度 ..........49
18. 統計分析 ..........50
伍、結果..........51
一、攝護腺癌細胞中 HMGB1 基因弱化 (Knockdown) 細胞株篩選.... 51
1. DU145 及 PC3 攝護腺癌細胞株 HMGB1 基因弱化結果........51
2. DU145 攝護腺癌細胞株之 HMGB1 基因弱化穩定性 ...........51
二、HMGB1 表現量對於攝護腺癌之 EMT 能力相關特性之探討 .52
1. HMGB1 基因弱化對於攝護腺癌中 HMGB1 釋放之調控 .....52
2. HMGB1 表現量對於攝護腺癌胞內鈣離子濃度之調控 ...........52
3. HMGB1 表現量對於攝護腺癌細胞 NF-κB 訊息傳遞路徑之調控 .... 52
4.Ethyl pyruvate 對 HMGB1 誘導之 DU145 細胞移性之影響 ....53
5.HMGB1 表現量對於攝護腺癌細胞在 poly-HEMA 處理環境下黏附能力影響 ..........53
6. HMGB1 表現量對攝護腺癌細胞在 E-cadherin 及 vimintin 表現調控之影響..........54
7. HMGB1 表現量對於攝護腺癌細胞之 E-cadherin 與 vinmintin螢光表現之影響..........54
8. HMGB1 表現量對於攝護腺癌細胞 snail 轉位活性之調控......55
9.HMGB1 表現量對於攝護腺癌細胞 GSK-3β 磷酸化調控之影響..... 55
10. HMGB1 表現量對於攝護腺癌細胞之 snail 表現量之調控..56
11. HMGB1 表現量差異對於攝護腺癌 snail 穩定性及 E-cadherin表現量間之調控..... 56
12. HMGB1 經 PI3K 及 MAPK 訊息傳遞路徑調控攝護腺癌細胞 EMT 之影響 ...... 57
13. HMGB1 表現量調控攝護腺癌細胞 TGF-β 之表現量。 .......57
14. HMGB1 經 Rac1/cdc42 訊息傳遞路徑調控攝護腺癌細胞 EMT之影響 ..... 58
15. HMGB1 表現調控攝護腺癌細胞 EMT 相關發炎因子之表現量........ 58
三、HMGB1 表現對於攝護腺癌於無血清環境下對 EMT 能力相關特性之探討 ...... 59
1. 無血清環境下 HMGB1 對於攝護腺癌細胞 E-cadherin 及β-catenin 基因表現之調控..........59
2. HMGB1 基因弱化對於攝護腺癌在無血清環境下 HMGB1 釋放之調控 ...... 59
3. HMGB1 表現量對於攝護腺癌細胞在低血清誘導下移行能力之影響 ...... 60
4. HMGB1 表現量對於攝護腺癌細胞在無血清移行能力之影響 60
5. HMGB1 表現量對攝護腺癌細胞在無血清環境下 E-cadherin與 vimintin 表現量之調控 ........ 61
6. HMGB1 表現量對攝護腺癌細胞在無血清環境下 snail 轉位活性之調控 ..... 61
7. HMGB1 表現對於攝護腺癌細胞在無血清環境下 Rac1/Cdc42 -GSK-3β 訊息傳遞路徑之調控 ..........62
8. HMGB1 表現量於無血清環境下調控攝護腺癌細胞 EMT 相關發炎因子之表現量..........62
四、甘草甜素介入對於攝護腺癌細胞 EMT 能力之影響 ..........63
1. 甘草甜素對 DU145 parental 及 DU145 HMGB1-L 攝護腺癌細胞之細胞毒性影響..........63
2. 甘草甜素對不同 HMGB1 表現量之攝護腺癌細胞 E-cadherin表現量之影響....63
3. 甘草甜素對不同 HMGB1 表現量之攝護腺癌細胞移行能力之影響..........63
陸、討論..........65
柒、結論..........76
參考文獻..........10
Bioactive food components alter one-carbon metabolic kinetics in vivo
No full textBackground Bioactive food components are nonessential
nutrients that are present in foods; many of them have been found to elicit the capacity to promote human health by modulation of one or more metabolic processes. The objective of this study was to examine the regulatory effects of specific bioactive food component supplementation on the protein abundance, gene expression and the activity of the key enzymes involved in adoMet homeostasis. Study design Study 1 was conducted to investigate the impacts of compound A supplementation on one-carbon kinetics in mice at young age. Study 2 was conducted to investigate the effects of parental compound A supplementation (during mating, gestation and lactation) on one-carbon metabolism in the offspring. In study 1, twelve male C57BL/6J mice aged 3 weeks were randomly assigned to receive either the control (CTL) or the compound A supplemented diet for 4 weeks. In Study 2, all parental mice received the compound A supplemented during mating, gestation and lactation. After weaning, the littermates continually received either the control diet or the compound A supplemented diet for an additional period of 4 weeks. Result. Compound A supplementation at a young age but not during gestation altered the hepatic and whole blood S-adenosylmethionine homeostasis. The isotopic tracer studies indicated that compound A supplementation tended to promote homocysteine remethylation fluxes in the tissues. Conclusion. The present study gives us new insights into the effects of compound A supplementation on homocysteine metabolism.背景 先前文獻及本研究室先前研究顯示食品中有
些活性物質會改變體內同半胱胺酸或硫-腺核?甲硫胺酸的含量。前者為心血管疾病的指標，後者為體內的甲基提供者，兩者皆為單碳代謝途徑中的關鍵代謝物，已知單碳代謝生化路徑的改變與許多人類病理狀態有關。本論文以動物模型探討不同的食品活性物質對於單碳代謝的影響。實驗設計 建立兩種動物模型，分析複合物A對S-腺?基甲硫氨酸代謝途徑以及其相關代謝產物如S-腺核?同半胱胺酸、同半胱胺酸及其相關代謝酵素活性、代謝酵素蛋白和基因表現量之影響。此外以同位素標定的方法去探討體內甲基代謝相關路徑分流的變化。實驗設計1為當代研究，探討早期餵食四週複合物A對當代老鼠代謝之影響。以複合物A混入飼料給予餵食四週後犧牲、取得各組織分析。實驗2為跨代研究，探討親代在交配、懷孕及哺乳期間補充上述複合物A對其子代代謝之影響。親代公母鼠在交配懷孕哺乳期間均攝食複合物A補充之飼料，子代斷奶後繼續餵食複合物A補充隻飼料四週進行分析。結果 在動物模型中，複合物A補充對當代及子代有不同影響。在當代鼠研究中,肝臟中腺?基甲硫氨酸增加,同時肝臟中腺?同半胱胺酸顯著性地下降。然而在子代鼠並未見到類似的情況。兩種動物模型中複合物A在不同組織中代謝路徑則較類似。複合物A會促進當代及子代肝臟、骨髓及大腦中同半胱胺酸再甲基化路徑。然而當代鼠主要顯現出較低腺?同半胱胺酸，而子代老鼠則在血漿中的同半胱胺酸有顯著減少現象。結論 當代及跨代餵食複合物A可透過改變肝臟及血液中的甲硫氨酸腺?基?移?及其他特定基因表現，進而改變相關代謝產物和代謝途徑。中文摘要-----------------------------------------------------------------------------------------iii
Abstract-----------------------------------------------------------------------------------------iv
Introduction-------------------------------------------------------------------------------------1
Materials and Methods----------------------------------------------------------------------------3
Results------------------------------------------------------------------------------------------14
Discussions--------------------------------------------------------------------------------------26
Figures------------------------------------------------------------------------------------------34
Tables-------------------------------------------------------------------------------------------38
References---------------------------------------------------------------------------------------7
ATP itself changes the protein folding/assembly of Escherichia coli uridine phosphorylase
No full textATP is an important metabolite found abundantly in cells. Except being as energy source, it was recently found that ATP also plays as a chemical chaperone to facilitate the folding of an important enzyme, Escherichia coli glyceraldehyde-3-phosphate dehydrogenase. This novel function of ATP is granted by the specific interaction to partially unfolded protein conformation. However, it is still unclear if this chaperone role is unique between ATP and Escherichia coli glyceraldehyde-3-phosphate dehydrogenase. To answer this question, we investigated the interaction between ATP and another protein, Escherichia coli uridine phosphorylase.
Unfolding equilibrium of this protein was measured by pulse proteolysis and tryptophan fluorescence. The results both showed the same apparently destabilizing effect in the presence of 3 mM ATP. Interestingly, ATP decreased Cm value with two transitions that are only monitored by tryptophan fluorescence. This difference compared to pulse proteolysis suggests the possible change of protein quaternary structure. The folding and unfolding kinetics of this protein were also determined. The results show that ATP only accelerates the unfolding but folding rates. This result was confirmed with the activity assay during the refolding of uridine phosphorylase. Moreover, the specificity test also showed decreased stability in the presence of other nucleotides.
In summary, ATP plays a different role on the folding of Escherichia coli uridine phosphorylase. This interaction may be significant to the change of turnover rate or function of this protein.ATP是細胞內普遍存在的重要代謝產物，除了作為細胞的能量來源外，在最近一篇大腸桿菌甘油醛3-磷酸脫氫酶 (glyceraldehyde-3-phosphate dehydrogenase，GAPDH) 的折疊與組合研究中，發現ATP可以加速該蛋白質的折疊速度，因此在GAPDH的蛋白質折疊過程中扮演著類似於chemical chaperone的角色。此項研究成果賦予了ATP全新的生物體功能，並同時證明了小分子對於蛋白質的作用不是只限定在完全折疊好狀態 (folded state) 的蛋白質。然而，ATP於GAPDH中所扮演的功能(chemical chaperone) 是否為唯一的特例，我們仍然不清楚。因此，我們進一步研究了ATP與大腸桿菌尿苷磷酸化酶 (uridine phosphorylase，UPase) 間的作用。
首先，我們分別用Pulse Proteolysis與螢光光譜儀進行了蛋白質熱力學的穩定度實驗，兩者的實驗結果均顯示3 mM ATP可以明顯地降低此酵素的穩定度。有趣的是，螢光光譜儀的觀察結果指出ATP對UPase的去穩定化出現了兩個階段的轉變過程，該結果顯示UPase受到ATP的影響可能發生了四級結構的轉變。再者，我們也進行了UPase的展開動力學 (unfolding kinetics) 與折疊動力學 (folding kinetics) 實驗，結果顯示ATP僅可以增加UPase展開的速度，但對於折疊速度卻沒有影響，而同樣的結果也由後續UPase酵素活性恢復的實驗結果所應證；最後，在UPase與ATP結合的專一性測試中，我們發現了數種核苷酸皆對於UPase有同樣去穩定化的情形。
相較於GAPDH，ATP在UPase的折疊中扮演著不一樣的角色，而這樣不一樣的作用方式，可能與UPase在細胞中的轉換速率 (turnover rate) 或生理功能的調節有關。中文摘要 Ⅱ
英文摘要 Ⅲ
目次 Ⅳ
圖目次 Ⅶ
表目次 Ⅷ
第一章 前言 1
一、 蛋白質的重要性 1
（一） 生理功能與折疊 1
（二） 可能影響正確折疊的因子 1
（三） 小結 2
（四） 研究動機 2
二、 蛋白質折疊 2
（一） 折疊過程中的不同狀態 2
（二） 穩定度 2
（三） Pulse Proteolysis 3
（四） 小分子對蛋白質的影響 3
（五） 小結 4
三、 假說（Hypothesis） 4
四、 大腸桿菌尿苷磷酸化酶（Uridine Phosphorylase，UPase） 5
五、 研究簡述 6
（一） 流程 6
（二） 方法 6
（三） 結果 6
第二章 材料與方法 7
一、 UPase之蛋白質表現及純化 7
（一） 純化pET21a質體 7
（二） 聚合酶連鎖反應放大UPase基因 7
（三） 基因接入質體 9
（四） 大腸桿菌BL21（DE3）中大量表現UPase 10
（五） UPase純化 11
（六） 聚丙烯醯胺膠體電泳（SDS-PAGE） 14
二、 蛋白質折疊熱力學 15
（一） 螢光光譜法 15
（二） Pulse Proteolysis 18
三、 蛋白質折疊動力學 20
（一） 展開動力學（Unfolding Kinetics） 20
（二） 折疊動力學（Folding Kinetics） 21
四、 酵素活性 22
五、 分子篩層析（Size Exclusion Chromatography，SEC） 23
六、 其它小分子對於蛋白質的作用 24
第三章 結果與討論 25
一、 蛋白質表現與純化 25
（一） 基因選殖 25
（二） 蛋白質純化 27
（三） 小結 28
二、 蛋白質折疊熱力學 28
（一） 利用Pulse Proteolysis測定Unfolding Equilibrium 28
（二） 利用螢光光譜儀測定Unfolding Equilibrium 29
（三） 討論 30
（四） 小結 31
三、 蛋白質折疊動力學 31
（一） 展開動力學（Unfolding Kinetics） 31
（二） 折疊動力學（Folding Kinetics） 32
（三） 討論 32
（四） 小結 34
四、 酵素活性 34
（一） 標準曲線 34
（二） 活性恢復結果 35
（三） 討論 35
（四） 小結 35
五、 四級結構變化 36
（一） 標準曲線 36
（二） 分子篩層析：UPase 36
（三） 分子篩層析：His Taq UPase 38
（四） 小結 38
六、 ATP專一性 38
（一） 比較：ATP、ADP、AMP、Adenosine 39
（二） 比較：ATP、dATP 39
（三） 比較：ATP、UTP、CTP、GTP 39
（四） 比較：Uridine、Phosphate、5-FU 40
（五） K+ 40
（六） 小結 40
第四章 結論 41
第五章 未來展望 42
第六章 參考文獻 43
圖 47
表 74
附錄 8
The microencapsulation of Lactobacillus plantarum by alginate-gelatin complex coacervation
No full textThe Lactobacillus plantarum is Gram-positive facultatively anaerobic bacteria, which are common in the intestinal tract of humans. They are important in promoting and maintaining human health. In the present study, using alginate and gelatin by complex coacervation to achieve probiotic encapsulation. Lactobacillus plantarum were encapsulated in alginate—gelatin gels. The main goal of the study was to characterize the resistance of the target strain in alginate-gelatin coacervates processed by freeze-drying, the encapsulate and the release behaviors under simulated gastrointestinal tract conditions to be able to assess the feasibility as an delivery system for probiotics. In addition, the cell viable of freeze dried capsules at room temperature and at 4℃ for eight weeks was detected. We found that coacervated capsules were shown to be more resistance to freeze-drying and simulated gastrointestinal conditions than free ones and single formula, specially under the conditions the ratio of protein and polysaccharide was 4：1. The encapsulation efficiency of alginate–gelatin complex coacervation was achieved 72%. The encapsulated L. plantarum maintained its viability for a longer shelf life at 4℃ than at RT during the storage time.植物乳桿菌(Lactobacillus plantarum)為兼性厭氧桿狀的革蘭氏陽性菌，普遍存在於人類的腸道中，於促進及保持人類身體健康上扮演很重要的角色。本研究中利用褐藻酸鈉及明膠藉由複合凝聚(Complex coacervation)進行益生菌的微膠囊化(Probiotic encapsulation)，將植物乳桿菌(Lactobacillus plantarum)包覆於複合凝聚的膠體。研究的主要目的為瞭解目標菌株在褐藻酸鈉–明膠複合凝聚體中的包埋情形和冷凍乾燥處理後的抗性及在模擬胃腸道中釋放行為的特質，藉此評估利用此包埋方法作為益生菌在體內運送系統的可行性。另外，也偵測冷凍乾燥後包埋菌體的膠囊在室溫及4℃兩個不同溫度下八周儲存時間的菌體存活率。研究結果顯示褐藻酸鈉–明膠在pH 4.5–3.0之間可形成複合凝聚，菌體包埋在複合凝聚膠體比在游離態及單一配方的組別對冷凍乾燥處理和模擬胃腸液環境的耐受性更高，尤其是在蛋白質和多醣的比例為4：1的條件下。褐藻酸鈉–明膠凝聚體的包覆率高達72%。經包覆的菌體有較長的貯藏期限且在4℃下的存活率比在室溫來得高。謝誌 i
中文摘要 ii
Abstract iii
目次 iv
圖目次 viii
表目次 x
第一章 前言 1
第二章 文獻回顧 4
2.1. 益生菌之簡介 4
2.2. 乳酸菌之簡介 5
2.2.1. 乳酸菌作為益生菌應具備的條件 6
2.2.2. 乳酸菌的益生功效 7
2.2.2.1 抗過敏作用 7
2.2.2.2. 抗癌功效 9
2.2.2.3. 緩和乳糖不耐症 (Lactose intolerance) 9
2.2.2.4. 抗氧化功效 10
2.2.2.5. 合成維生素及生物活性物質 10
2.2.2.6. 降低血清中的膽固醇 10
2.2.2.7. 降低高血壓 11
2.2.2.8. 乳酸菌應用於腸道內微生物平衡的調節 12
2.2.3. 植物乳桿菌之介紹 13
2.3. 益生菌的膠囊化 17
2.3.1. 微膠囊化技術簡介 18
2.3.1.1. 膠囊化處理 18
2.3.1.2. 乾燥處理 22
2.4. 微膠囊化技術使用之生材 31
2.4.1. 褐藻酸鈉(Alginate) 31
2.4.2. 明膠(gelatin) 34
第三章 研究目的 37
3.1. 目的 37
3.2. 實驗架構圖 37
第四章 材料與方法 39
4.1. 材料與設備 39
4.2. 微生物培養 41
4.3. 微生物生長pH值測定 41
4.4. 褐藻酸鈉—明膠微膠囊化製備 42
4.5. 濁度分析 43
4.6. 包覆效率測量 43
4.7. 冷凍乾燥存活率評估 44
4.8. 模擬胃腸道耐受性試驗 45
4.9. 數位顯微鏡觀察 45
4.10. 掃描式電子顯微鏡觀察 46
4.11. 樣品儲存穩定性試驗 46
4.12. 統計分析 46
第五章 結果與討論 47
5.1. 微生物培養 47
5.2. 不同pH值條件下的複合凝聚程度與對菌體的包覆情形及影響 47
5.3. 褐藻酸鈉–明膠之複合凝聚對L. plantarum的保護作用 57
5.4. 型態觀察 70
5.5 褐藻酸鈉及明膠及其複合凝聚包覆之L. plantarum之儲存安定性 70
第六章 結論 78
第七章 參考文獻 7
Study on the relationship between the chemical composition of beer and astringency perception
No full textBeer was one of the ancient beverages that could be dated back to Mesopotamia. The beer kinds were various due to the development of crafts and the usage of different material. The flavor and taste of the beer would influence the acceptability of customers toward a new kind of beer, and astringency might be one of this unfavorable taste.
This research indicated that the content of tyrosine and tryptophan seemed to be unchanged during brewing. The variation of the phenolic compounds just coincided with the mode of the alcohol production, sugar and amino acid utilization. The content of p-coumaric acid, trans-ferulic acid and vanillic acid compared to other phenolic compounds varied during main fermentation, and the content of phenolic compounds were primarily determined in this stage. Vanillic acid , trans-ferulic acid and p-coumaric acid were related with the formation of volatile phenolic flavor. There was a highly positive correlation between trans-ferulic acid and p-coumaric acid , and it might attribute to the similar metabolic pathway of yeast.
There was a moderately positive correlation between astringency and tannin content, its correlation coefficient fell between 0.618 and 0.730. The relationship could be observed in the sample with higher alcohol content (5.0-5.7%), lower sugar content, and tannin content above 0.40 g/L.
In general, there was a moderately positive correlation between astringency and lingering bitterness. According to the principal component analysis chart, procyanidin B2 in beer have positive effect on beer body and enrich the beer.
We observed that total acidity of beer also influenced the astringency perception of beer. Rutin, an important astringency substance found in wine, was also correlated with astringency perception in beer. The astringency caused by this compound was primarily observed in the sample with lingering bitterness, lower alcohol and sugar content.啤酒可追溯至美索不達米亞時代，為古老酒精飲料之一。隨著各式釀造工藝發展與不同原料的使用，使得啤酒種類益發多元。啤酒香氣與口味每每影響消費者對新啤酒接受度，澀味便屬於啤酒中不良風味的一員。
本研究顯示啤酒釀造過程中，胺基酸利用於主酵期(0-5天)較為明顯，但明顯不同胺基酸酵母利用率有相當大之差異。酪胺酸與色胺酸在整個發酵過程中幾乎不產生變化，一開始發酵半胱胺酸、絲胺酸、離胺酸及精胺酸利用速度最快，應為酵母之必須胺基酸。酚類在發酵過程中變化趨勢恰與酵母活動酒精生成、降糖及胺基酸變化相符，其中對-香豆酸、反-阿魏酸及香草酸於主發酵期間有較大增減趨勢，啤酒成品中酚類含量大致於主酵期結束後即決定，大部份酚酸與胺基酸於升溫還原期含量大致上已穩定，但香草酸、對-香豆酸及反式-阿魏酸於此階段仍有明顯波動，這部分與風味物質，一些揮發性酚類之產生相關。在酵母活動中啤酒中反-阿魏酸含量與對-香豆酸含量彼此相關性達0.85以上，前者含量多時，後者含量必定較高。
澀味與單寧含量具中度相關性，相關性為0.618-0.730，於酒精度介於5.0-5.7%左右，且糖度低，單寧含量大於0.40 g/L之品項中相關性愈明顯，澀味與不悅後苦普遍存在中度正相關。由品評酒主成分分析圖顯示，啤酒中原花青素B2對整體酒質有正面效果，其存在使酒體更為豐富。
單寧於啤酒亦與不悅後苦有關聯，當酒精度高於5.0%時，單寧含量高者致使不悅後苦感受也明顯。澀味、不悅後苦及不悅酸感三者皆會影響啤酒整體品質，也有彼此凸顯情形，品評結果顯示前兩者有中度正相關。總酸含量則較不悅酸感此項目更具影響，總酸含量高，容易感到有澀味，這很有可能因為酸具有揮發性，不悅酸感綜合氣味與口味。紅酒中相當重要的澀味物質，芸香素(rutin)於啤酒中與澀味也有關，主要與糖度1.0 plato左右，酒精度偏低，後苦明顯的酒關聯較大。目錄
摘要 i
Abstract ii
圖次目錄 v
表次目錄 vii
前言 1
壹、文獻回顧 2
一、酚類物質簡介 2
(一) 麥芽來源 2
(二) 啤酒花來源 4
(三) 植物來源及酵母代謝 9
二、啤酒製程簡介及過去釀酒工藝題及澀味問題 13
三、澀味之研究 19
貳、實驗架構 26
參、材料與方法 27
一、實驗材料 27
(一) 原料 27
(二) 試藥 29
(三) 儀器設備 31
二、實驗方法 31
(一) 樣品製備 31
(二) 啤酒化學成分分析 32
1. 總酸 32
2. 總多酚 33
3. 苦味度 33
4. 總單寧 33
5. 游離胺基氮 33
6. 胺基酸 34
7. 以LC/MS/MS分析酚酸及多酚類 35
8. 由麥殼中分離澀味物質-大麥芽胍鹼(hordatine) 35
(三) 啤酒一般性理化分析 36
1. 酒精度及糖度 36
2. pH 36
(四)感官品評 36
1. 品評員篩選 36
2. 品評員訓練 36
3. 樣品品評 37
(五)統計方法 37
1. 圖奇氏檢定(tukey's test) 37
2. 主成份分析(PCA) 37
肆、結果與討論 39
一、由麥殼萃取物製備大麥芽胍鹼(Hordatine) 39
二、啤酒之化學成份分析 42
1. 單寧 45
2. 以LC/MS/MS分析酚酸及多酚類 46
三、發酵中各成份消長趨勢 48
四、啤酒之一般性理化成份分析 67
五、LC-MS/MS分析結果 70
六、感官品評數據結合進行主成份分析(PCA)結果 85
伍、結論 98
陸、參考文獻 99
柒、附錄 10
Research on Mechanism of Probiotic Actions on Tolerance Induction Towards Pathogen-induced Hepatocytic Inflammation
No full textPathogen peptidoglycans (PGN) or lipopolysaccharides are detected via host's innate immune system. This course of action is executed by toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) proteins and peptidoglycan recognition proteins (PGLYRPs), which have been identified relaying signals to nuclear factor-kappa B (NF-κB), a transcription factor leading to pro-inflammatory cytokine production. In this study, seven strains of probiotics were first processed into crude cell wall extracts, followed by testing if crude cell wall extracts of probiotics could lessen pro-inflammatory processes launched by Escherichia coli or Staphylococcus aureus crude cell wall extracts.
The results showed that prior exposure of HepG2 cells to probiotic crude cell wall extracts render them desensitized to subsequent E. coli or S. aureus challenge. We proposed a new mechanism for which probiotic crude cell wall extracts attenuate inflammation induced by E. coli is because of the activation of TOLLIP, a negative regulator of TLR4, while attenuate S. aureus-induced inflammation is due to the triggering of PGLYRP3, which serves as an anti-inflammation mediator, counteracting the function of PGLYRP2 and NOD2.
In addition to providing new perspectives for illuminating the mechanisms underlying anti-inflammatory effects of probiotics, results of this work may help establish an approach for selecting hepatoprotective probiotic strains.病原菌肽聚醣或脂多醣是經由宿主的先天免疫系統辨認。這項辨認行動是由類鐸受體（TLRs），核苷酸結合寡聚化結構域（NODs）蛋白和肽聚醣識別蛋白（PGLYRPs）所執行，這些受體已經被證實會傳遞信號給NF-κB，一種轉錄因子，導致促炎細胞因子的產生。在本研究中，七株益生菌被事先加工成粗細胞壁提取物，以測試益生菌粗細胞壁提取物是否能降低由大腸桿菌或金黃色葡萄球菌粗細胞壁提取物發動的促炎症過程。
結果顯示， HepG2細胞預先暴露於益生菌粗細胞壁提取物使其對於隨後大腸桿菌或金黃色葡萄球菌刺激不若原先敏感。我們提出一個新的機制解釋益生菌粗細胞壁提取物降低大腸桿菌誘導炎症是因為活化TOLLIP，一種類鐸受體的負調節控制蛋白，而降低金黃色葡萄球菌誘導的炎症是由於活化PGLYRP3，作為抗發炎媒介，抵消PGLYRP2和NOD2的功能。
除了闡釋益生菌抗炎作用機制的新觀點，這項工作的成果可能有助於建立護肝益生菌菌株的篩選方法Acknowledgements……………………………………………………i
Chinese abstract………………………………………………………ii
English abstract……………………………………………………iii
Contents………………………………………………………………iv
Contents of appendices………………………………………………vi
Contents of tables…………………………………………………vii
Contents of figures………………………………………………viii
Introduction……………………………………………………………1
Material and methods………………………………………………8
Results…………………………………………………………………12
Discussion………………………………………………………………18
References………………………………………………………………23
Appendices……………………………………………………………30
Tables…………………………………………………………………32
Figures…………………………………………………………………3