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Field Study of Performance and Effectiveness on Negative-Pressured Isolation Rooms during Operation
No full text誌謝 I
中文摘要 II
英文摘要 IV
第一章 前言 1
1-1研究背景 1
1-2研究目的 3
第二章 文獻回顧 4
2-1負壓隔離病房定義與應用 4
2-2負壓隔離病房壓差值之設計 11
2-3負壓隔離病房之空調規劃 14
2-4負壓隔離病房之安全管制與防護具使用 16
2-5負壓隔離病房性能規範 17
第三章 材料與方法 19
3-1實驗場所 21
3-2量測儀器介紹 22
3-3現場負壓隔離病房性能評估方式 32
第四章 結果與討論 35
4-1檢測結果 40
4-1-1 A醫院負壓隔離病房檢測結果: 40
4-1-2 B醫院負壓隔離病房檢測結果: 41
4-1-3 C醫院負壓隔離病房檢測結果: 42
4-1-4 D醫院負壓隔離病房檢測結果: 43
4-2綜合討論 44
4-2-1定性檢測討論 44
4-2-2定量檢測討論 44
第五章 結論與建議 46
5-1結論 46
5-2建議 46
參考文獻 48[[abstract]]本研究針對4家醫院共52間負壓隔離病房分別進行定性與定量檢測，以評估其運作效能。並依據我國「負壓隔離病房作業參考手冊」規定，進行可行改善方案探討，以提供未來改善或設計負壓隔離病房的參考。負壓隔離病房現場性能檢測分為定性與定量測試兩種方式。定性測試為藉由發煙管產生之白色煙霧，來觀察氣流方向；定量測試則是以輪葉式風速計測定進氣口或排氣口的平均風速後，搭配開口面積獲得進氣或排氣風量；或直接利用微壓風量風速計量測進氣風量。以雷射測距儀測量病房尺寸並計算其空間體積後，則可以此空間體積結果與前述量測之進氣風量，評估每小時換氣次數(ACH)，最後再利用微壓校正儀量測走廊與病房兩空間的壓差值。本研究以氣流自走廊流向病房，每小時換氣次數達8次以上且壓差值達到8Pa做為合格的判定依據。
根據檢測結果發現，定性測試合格率為100%，而定量測試合格率為34.6% (18/52)。其中定量測試不合格的原因以每小時換氣次數未達8ACH以上者佔59.6% (31/52)，壓差值未達8Pa以上者則佔13.4% (7/52)。針對定量測試每小時換氣次數未達8ACH以上者，可同時增加病房進排氣量，以達到8ACH以上並維持壓差值達到8Pa以上的要求；此外亦可藉減少病房體積以提升每小時換氣次數至≧8ACH來進行改善。如果每小時換氣次數已達到8ACH以上的要求，但壓差值未達8Pa以上者，則先確認病房氣密性後，利用調整門縫開口大小或增加排氣量，以符合壓差值要求。
There are 52 negative pressured isolation rooms (NPIRs) in four different hospitals were assessed by both qualitative and quantitative testing, to evaluate the performance during operation. To improve the operating performance and effectiveness, feasible solution was then proposed based on “Negative pressured isolation room reference manual of CDC and IOSH (NPIRRM)” in Taiwan. Field study of performance and effectiveness in NPIRs were divided into qualitative and quantitative testing. The smoke tube was used for the observation of airflow pattern in qualitative testing. In quantitative testing, anemometer was utilized to measure air velocity as well as intake or exhaust air volume if the opening area is available. The air-change rate (ACH) was then obtained by intake air volume and room volume. Finally, the micromanometer was used to measure pressure differential of NPIRs.
The results of qualitative analysis in 52 NPIRs indicate that all airflow directions were correct. The measurements of air-change rate (ACH) less than 8ACH are approximate to 59.6% (31/52). The pressure differential of studied NPIRs was less than 8Pa which suggested in the NPIRRM of Taiwan about 13.4% (7/52). Therefore, based on criteria of 8ACH and 8Pa in quantitative testing, only 34.6% (18/52) meet the requirements indicated in NPIRRM of Taiwan. For those NPIRs with air-change rate less than 8ACH, increasing intake and exhaust air volume at the same time was suggested to improve air change rate and maintain the pressure differential. In addition, downgrade of the room volume is also a possible way to meet the requirements. NPIRs with pressure differential smaller than 8Pa, which air-tightness should be confirmed, and then decreasing doorway and increasing exhaust air volume are adjusted to meet the above requirements
Purification And Characterization Of A Bacteriocin Produced By Lactic Acid Bacteria
No full text目 錄
授 權..........................................................................................................i
誌謝............................................................................................................ii
目 錄.........................................................................................................iii
中文摘要...................................................................................................iv
英文摘要.....................................................................................................v
壹、導論.......................................................................................................1
貳、研究目的............................................................................................18
參、材料與方法.........................................................................................19
肆、結果與討論........................................................................................25
伍、結論...................................................................................................29
陸、參考文獻...........................................................................................30
柒、圖........................................................................................................50
捌、表...................................................................................................83[[abstract]]細菌素是由細菌所產生具有抑菌效果的蛋白質，可以殺菌或抑制其他細菌生長，目前被認為在食品工業上極具發展潛力。本實驗室自海洋環境（如各種貝類:蛤仔、牡蠣。魚類及蝦類腸道等）中篩選得到6株乳酸菌株，並與本實驗室菌種庫（共250隻菌株）比較抑菌活性。結果發現A菌株與B菌株所產之細菌素具有較顯著的抑菌能力，可抑制水產病原菌Vibrio alginolyticus 、Vibrio parahaemolyticus，因此選擇作為本研究之篩選菌株。根據生化測試及16S rDNA 序列分析比對結果，初步鑑定菌株A、B皆屬於L.plantarum。以Agar well difusion抑菌結果顯示A菌株於發酵48小時後經硫酸銨沉澱(ammonium sulfate)所得的粗萃取液對病原菌Vibrio alginolyticus有最佳的效果（B菌株為60小時），而Vibrio parahaemolyticus則是發酵72小時後的粗萃取液有較佳的效果（B菌株為48小時）。進一步將A菌株的粗萃取液以FPLC膠體過濾層析分離後進行抑菌試驗，結果顯示對Vibrio alginolyticus具有抑制效果，顯示菌株A所產生的細菌素對水產病原菌有抑制作用，對水產養殖上的應用具相當潛力。
Bacteriocins are proteinaceous toxins produced by bacteria to kill or inhibit the growth of other bacterial strains. They are considered having potential in the application of the food industry. We screened the antibacterial activity of six lactic acid bacteria strains from the marine environment (such as a variety of shellfish: clams, oysters, fish and shrimp gut, etc.) and our culture collections (250 strains). The results showed that the bacteriocins produced by strain A and B have significant antimicrobial activity and inhibit aquatic pathogen of Vibrio alginolyticus and Vibrio parahaemolyticus strains.Therefore, we chose strain A and B as the screening of this study. Based on biochemical tests and 16S rDNA sequence analysis, the preliminary identification of strains A and B belong to L. plantarum. Agar well diffusion antibacterial results show that the crude extracts with ammonium sulfate precipitation of strain A after 48 hours fermentation effectively inhibit Vibrio alginolyticus, and that after 72 hours fermentations to inhibit Vibrio parahaemolyticus. In strain B, the crude extracts with ammonium sulfate precipitation of 60 hours fermentation effectively inhibit Vibrio alginolyticus, and that of 48 hours fermentations to inhibit Vibrio parahaemolyticus. Further, the antibacterial activity measured by the crude extract of strain A fermentation after FPLC gel filtration separation. The results showed that the bacteriocin produced by strain A has an inhibitory effect of Vibrio alginolyticus, should be considered to have considerable potential for inhibiting aquatic pathogens on aquaculture applications
Characteristic Analysis Of Tannases From Various Functional Microorganisms
Get PDF致謝………………………………………………………………………………………………………………………………………………………………i
中文摘要………………………………………………………………………………………………………………………………………………………ii
英文摘要………………………………………………………………………………………………………………………………………………………iv
目錄………………………………………………………………………………………………………………………………………………………………vi
圖表目錄………………………………………………………………………………………………………………………………………………………viii
一、前言………………………………………………………………………………………………………………………………………………………1
1.茶
2.單寧(Tannin)
3.單寧醯基水解酶(Tannin acyl hydrolase)
4.沒食子酸(Gallic acid)
5.Eurotium cristatum
6.Penicillium citrinum
7.Fusarium solani
二、研究動機與目的…………………………………………………………………………………………………………………………………9
三、實驗材料………………………………………………………………………………………………………………………………………………11
1.藥品及器材
2.儀器
四、實驗方法………………………………………………………………………………………………………………………………………………14
1.機能性微生物菌株培養及單寧酶的產生
2.培養基
3.生產單寧酶之平板篩選試驗
4.含單寧酶之培養液收集
5.以硫酸銨沉澱濃縮粗樣本
6.單寧酶酵素活性分析
7.發酵槽大量培養
8.Native Polyacrylamide gel preparation
9.銀染
10.單寧酶圖譜活性分析
11.Sodium Dodecyl Sulfate – Polyacrylamide gel electrophoresis
五、結果………………………………………………………………………………………………………………………………………………………23
1.測定各種機能性微生物分解單寧酸的能力
2.在平板篩選試驗分析各種機能性微生物之單寧酶活性
3.比較各種機能性微生物在培養基中產生的單寧酶活性
4.比較各種機能性微生物在不同培養基中產生的單寧酶活性
5.單寧酶的分離與純化
6.單寧酶胺基酸序列比對
六、討論………………………………………………………………………………………………………………………………………………………27
七、結論………………………………………………………………………………………………………………………………………………………30
參考文獻………………………………………………………………………………………………………………………………………………………32
圖表附錄………………………………………………………………………………………………………………………………………………………36[[abstract]]茶葉中含有單寧酸、咖啡因及茶鹼，對身體會造成負面的影響，但也常常被忽視。許多科學文獻報告也證實，利用生物技術能夠減少茶葉中的單寧酸、咖啡因及茶鹼。「金花菌(Eurotium cristatum)」是中國普洱茶與茯磚茶中的珍寶。它能讓茶湯顏色更漂亮、口感更柔軟、甘醇。金花菌對人體降血脂、減肥、降三高、抑制癌細胞等功效均發表於諸多著名學術期刊及醫學臨床報告。
在台灣，我們研發團隊首先從台灣老烏龍茶葉中成功將「金花菌」分離篩選純化出來，經「食品工業研究所」菌種鑑定證實為「金花菌」，已成功開發低單寧酸、咖啡因及茶鹼並結合『台灣味的金花菌茶』之保健茶葉。
本研究中，我們進一步探討來自Eurotium cristatum、Penicillium citrinum及Fusarium solani之單寧酶特性。在單寧酶平板篩選試驗中，三者均有降解單寧之能力，但不同的菌株所降解的區域大小則不同，其中，以Eurotium cristatum生長的速率最慢，這可能與單寧酶之產量有關。
將Eurotium cristatum、Penicillium citrinum及Fusarium solani分別培養在添加2 %單寧酸的MSM中所收取之培養液進行單寧酶活性圖譜分析，結果顯示Penicillium citrinum及Fusarium solani有band出現，具單寧酶活性，Eurotium cristatum則無活性；若對照單寧酶活性定量分析實驗，則發現Penicillium citrinum及Fusarium solani粗樣本單寧酶活性高於Eurotium cristatum的粗樣本(七倍以上)，這可能是Eurotium cristatum在單寧酶圖譜活性定性實驗均無法顯現活性的原因；若將Eurotium cristatum、Penicillium citrinum及Fusarium solani分別培養在添加0.5 %單寧酸的MSM中所收取之培養液進行單寧酶活性圖譜分析，則全部無法顯現單寧酶活性，將各樣本進行單寧酶活性定量分析實驗後，發現培養於添加0.5 %單寧酸培養基樣本活性明顯較低。另外，單寧酶圖譜定性活性分析實驗中顯示，SDS與beta-mercaptoethanol會使Penicillium citrinum及Fusarium solani所生產之單寧酶失去活性。
未來將先進行單寧酶之純化，再利用LC/MS/MS及PCR方法選殖單寧酶cDNA，分析這些單寧酶胺基酸序列是否具有活性位置的高度保留序列，並將單寧酶的 cDNA序列轉殖至細菌或真菌之蛋白質表現系統，期望能夠透過這些表現系統產出大量具有活性之重組單寧酶。
Tea contains tannic acid, caffeine and theophylline ensure the body would be a negative impact, and is often ignored. Many scientific literature reports have also confirmed that the use of biotechnology can reduce the tannic acid, caffeine and theophylline in tea. “Eurotium cristatum”is the treasure in Pu-erh tea and Fuzhuan Brick tea in China. It can make tea color more beautiful, softer texture, and sweeter. Eurotium cristatum on hypolipidemic, weight-loss, reduction of three-high and inhibition of cancer cells effects have been published in many leading academic journals and medical clinical reports.
We first successfully isolated Eurotium cristatum from Taiwan Oolong tea and confirmed as the " Eurotium cristatum " by the strain identification from “Institute of Food Industry” in Taiwan. We have successfully developed low tannic acid, coffee and theophylline combined with an " Eurotium cristatum containing Taiwan health tea ".
In this study, we further investigated the characteristics of tannases from Eurotium cristatum, Penicillium citrinum and Fusarium solani. The results of tannin plate assay suggest that three of them all have ability in tannin-degradation and the zone sizes are different in different fungi. However, the growing rate of Eurotium cristatum was the most slowest. This is probably relative to production level of tannase.
We collected the secreted supernatant from the culture of Eurotium cristatum, Penicillium citrinum and Fusarium solani in MSM contain 2 % tannic acid and analyzed them by zymogram assay. The results showed that Penicillium citrinum and Fusarium solani have bands appeared with tannase activity but Eurotium cristatum revealed no activity. When compare the tannase activity from all crude samples analyzed between qualitative and quantitative assays, we found that the tannase activity from Penicillium citrinum or Fusarium solani is higher than Eurotium cristatum five times more by quantitative assay. It could be the reason why we can’t see any band appeared in all Eurotium cristatum zymogram assay. However, if we collected the secreted supernatant from the culture of Eurotium cristatum, Penicillium citrinum and Fusarium solani in MSM contain 0.5 % tannic acid and analyzed them by zymogram assay. They all showed no any bands appeared with tannase activity. All data demonstrated that crude samples from 0.5% tannic acid are lower than 2% tannic acid. Furthermore, we performed the addition of SDS or beta-mercaptoethanol in zymogram assay, they showed that the tannase activity was all abolished from Penicillium citrinum and Fusarium solani.
In the future, we will purify the tannase and further clone tannase cDNA by LC/MS/MS and PCR strategies. Then, we will try to align the amino acid sequences and found the conserve active site. We hope to construct the expression vectors in bacterial or fungi system and express large amount of recombinant tannase with high activity
The Modulation of Apoptosis by Green Tea Polyphenols in Prostate Cancer Cells
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中文摘要………………………………………………………………3
英文摘要………………………………………………………………4
致謝……………………………………………………………………5
第一章 緒論…………………………………………………………7
第一節、 前言……………………………………………………7
第二節、 實驗目的………………………………..……….……15
第二章 實驗材料與方法…………………………………….…..…16
第三章 實驗結果…………………………………………………...21
第四章 實驗討論與結論……………………………………………23
第五章 圖與表………………………………………………………25
第六章 參考文獻……………………………………………………32[[abstract]]近幾年因為平均壽命增加、飲食習慣改變及篩檢進步導致攝護腺癌之病例有快速增加的趨勢，攝護腺癌將來恐怕造成國人生命健康之重大威脅。綠茶多酚類化合物在過去研究指出證明具有抗氧化、抑制腫瘤血管新生、抗基因突變及抗腫瘤效能。本計劃以攝護腺癌之細胞株(LNCaP)進行體外培養，探討綠茶多酚類化合物對於細胞凋亡(apoptosis)訊息路徑的影響，我們將以流式細胞儀分析癌細胞之細胞週期、細胞凋亡以及使用西方墨點法分析細胞凋亡之相關訊息路徑變化情形與下游蛋白質的表現。以進一步探討綠茶多酚類化合物抑制攝護腺癌細胞之機轉。研究結果顯示，在添加不同濃度之EGCG (30、50、80μM)後明顯誘發細胞凋亡蛋白caspase3/7/8/9、parp、JNK等蛋白質之含量，亦可抑制細胞凋亡抑制蛋白BCL-2蛋白質之含量，且同時觀察到細胞凋亡蛋白Cyt-C含量亦明顯上升，綜合以上分子生物學細胞模式結果顯示，證實EGCG透過誘發細胞凋亡蛋白caspase、parp、JNK，抑制細胞凋亡抑制蛋白BCL並釋放細胞凋亡蛋白Cyt-C來達到誘發細胞凋亡反應的效果。本研究不止促進抗攝護腺癌細胞相關機制的瞭解，亦可開發出全新抗癌之替代療法，可作為臨床醫師之參考。目的希望可以大大降低本國花費於攝護腺癌上的醫療社會成本。
Prostate cancer is the most common cancer affecting men in Western countries. In Taiwan, it is the 5th common cancer in men and increasing incidence. Epigallocatechin-3-gallate (EGCG) represents the major Green Tea Polyphenol constituents, the most popular beverage next to water that has potential to be developed as a chemo preventive agent for prostate cancer. The effects of anti-prostate cancer by EGCG were never been systemic evaluated. The purpose of this study is to verify the inhibitory effect on prostate carcinogenicity including modulation of signal transduction of apoptosis by western blots and shift of cell cycle by FACscan flow cytometry. The mechanisms of anti prostate carcinogenesis, evaluation models will be established on EGCG. The results indicated that EGCG significantly induced the apoptosis protein levels of caspase3/7/8/9, parp and Cyt-C and JNK. In addition, and reduced the inhibitory protein levels of BCL-2. The apoptosis expression was significantly higher treated by EGCG than control. The results from these investigations will prove to be helpful for understanding the anti-prostate cancer mechanics by EGCG and provide novel insights in developing of chemopreventive agents in clinical medicines
Effects of single nucleotide polymorphism of MTERFD3 gene on mood disorders
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目錄-------------------------------------------------------------------------------------------i
圖表目錄----------------------------------------------------------------------------------------v
附錄-------------------------------------------------------------------------------------------vii
論文口試委員審定書-------------------------------------------------------------------------------viii
致謝--------------------------------------------------------------------------------------------ix
中文摘要-----------------------------------------------------------------------------------------1
英文摘要-----------------------------------------------------------------------------------------3
第一章 前言
第一節 情感性疾患(Mood disorder)-------------------------------------------------------------------5
第二節 憂鬱症(Depressive disorder)----------------------------------------------------------------5
第三節 躁鬱症(Manic depressive disorder, Bipolar (Affective) Disorder BPD)------------------------7
第四節 人類基因位點(SNP)與情感性疾患之間的相關性-------------------------------------------------------10
第五節 MTERFD3基因--------------------------------------------------------------------------------12
第六節 MTERFD3基因(SNP rs2287161)-----------------------------------------------------------------14
第七節 研究目的------------------------------------------------------------------------------------15
第二章 材料與方法
第一節 研究樣本------------------------------------------------------------------------------------16
第二節 組合國際診斷會談問卷(CIDI)--------------------------------------------------------------------16
第三節 基因分型SNP檢測------------------------------------------------------------------------------17
第四節 細胞培養------------------------------------------------------------------------------------18
第五節 建構MTERFD3表現質體--------------------------------------------------------------------------19
第六節 定點突變------------------------------------------------------------------------------------20
第七節 基因轉染------------------------------------------------------------------------------------22
第八節 啟動子分析----------------------------------------------------------------------------------23
第九節 統計方法------------------------------------------------------------------------------------24
第三章 結果
第一節 睡眠品質在不同組別受試者中有顯著差異-------------------------------------------------------------25
第二節 疾病診斷在不同基因型間的關係--------------------------------------------------------------------25
2.1 疾病診斷在不同基因型間統計分析--------------------------------------------------------------------25
2.2 匹茲堡睡眠品質量表總分在不同基因型與診斷的關係-------------------------------------------------------26
2.3 匹茲堡睡眠品質量表品質(Quality)在不同基因型與診斷的關係----------------------------------------------26
2.4 匹茲堡睡眠品質量表潛伏期(Latency)在不同基因型與診斷的關係--------------------------------------------27
2.5 匹茲堡睡眠品質量表時間(Duration)在不同基因型與診斷的關係---------------------------------------------27
2.6 匹茲堡睡眠品質量表效率(Efficiency)在不同基因型與診斷的關係-------------------------------------------28
2.7 匹茲堡睡眠品質量表困擾(Disturbances)在不同基因型與診斷的關係-----------------------------------------29
2.8 匹茲堡睡眠品質量表用藥(Medications)在不同基因型與診斷的關係------------------------------------------29
2.9匹茲堡睡眠品質量表白天功能(Dysfunction)在不同基因型與診斷的關係----------------------------------------30
第三節 MTERFD3 (rs2287161) SNP不同基因型對自殺的發生沒有影響--------------------------------------------30
3.1 合併病人組及健康組分析(Combine patient and control groups)----------------------------------------30
3.2 病人組分析(Patients)----------------------------------------------------------------------------31
3.3 健康組分析(Controls)----------------------------------------------------------------------------31
第四節 MTERFD3 (rs2287161) SNP不同基因型對焦慮的發生沒有影響---------------------------------------------32
4.1 合併病人組及健康組分析 (Combine patient and control groups)----------------------------------------32
4.2 病人組分析 (Patients)----------------------------------------------------------------------------32
4.3 健康組分析(Controls)-----------------------------------------------------------------------------33
第五節 MTERFD3 (rs2287161) SNP不同基因型對憂鬱的發生沒有影響----------------------------------------------33
5.1 合併病人組及健康組分析 (Combine patient and control groups)-----------------------------------------33
5.2 病人組分析(Patients)------------------------------------------------------------------------------34
5.3 健康組分析(Controls)------------------------------------------------------------------------------34
第六節 MTERFD3 (rs2287161) SNP會影響啟動子活性----------------------------------------------------------35
第四章 討論
第一節 睡眠品質量表在不同組別受試者中的差異-----------------------------------------------------------------36
1.1 匹茲堡睡眠品質量表總分在不同基因型與診斷的差異-----------------------------------------------------------36
1.2 匹茲堡睡眠品質量表品質(Quality)在不同基因型與診斷的關係--------------------------------------------------37
1.3 匹茲堡睡眠品質量表潛伏期(Latency)在不同基因型與診斷的關係------------------------------------------------37
1.4 匹茲堡睡眠品質量表潛伏期時間(Duration)在不同基因型與診斷的關係--------------------------------------------37
1.5 匹茲堡睡眠品質量表效率(Efficiency)在不同基因型與診斷的關係--37
1.6 匹茲堡睡眠品質量表困擾(Disturbances)在不同基因型與診斷的關係---------------------------------------------38
1.7 匹茲堡睡眠品質量表用藥(Medications)在不同基因型與診斷的關係----------------------------------------------38
1.8 匹茲堡睡眠品質量表白天功能(Dysfunction)在不同基因型與診斷的關係-------------------------------------------38
第二節 疾病診斷與情緒表徵在不同基因型間的關係----------------------------------------------------------------38
2.1 疾病診斷在不同基因型間統計分析的差異--------------------------------------------------------------------38
2.2 自殺傾向-------------------------------------------------------------------------------------------40
2.3 焦慮-----------------------------------------------------------------------------------------------40
2.4 憂鬱-----------------------------------------------------------------------------------------------41
第三節 rs2287161 SNP 會影響MTERFD3啟動子的活性------------------------------------------------------------41
第五章 結論---------------------------------------------------------------------------------------------43
參考文獻------------------------------------------------------------------------------------------------45
自述---------------------------------------------------------------------------------------------------89
圖表目錄
表1-1 匹茲堡睡眠品質量表在不同組別受試者中有顯著差異統計表------------------------------------------------------52
表1-2 匹茲堡睡眠品質量表總分和七項指標在不同基因型間的關係------------------------------------------------------53
表2-1 疾病診斷在不同基因型間的關係--------------------------------------------------------------------------55
表2-2-1 匹茲堡睡眠品質量表總分在不同基因型與診斷的關係(描述性統計) ----------------------------------------------56
表2-2-2 匹茲堡睡眠品質量表總分在不同基因型與診斷的關係(推論性統計) ----------------------------------------------56
表2-3-1 匹茲堡睡眠品質量表品質(quality)在不同基因型與診斷的關係(描述 性統計)-------------------------------------57
表2-3-2 匹茲堡睡眠品質量表品質(quality)在不同基因型與診斷的關係(推論性統計)--------------------------------------57
表2-4-1 匹茲堡睡眠品質量表潛伏期(latency)在不同基因型與診斷的關係(描述性統計)------------------------------------58
表2-4-2 匹茲堡睡眠品質量表潛伏期(latency)在不同基因型與診斷的關係(推論性統計)------------------------------------58
表2-5-1 匹茲堡睡眠品質量表時間(duration)在不同基因型與診斷的關係(描述性統計)-------------------------------------59
表2-5-2 匹茲堡睡眠品質量表時間(duration)在不同基因型與診斷的關係(推論性統計)-------------------------------------59
表2-6-1 匹茲堡睡眠品質量表效率(efficiency)在不同基因型與診斷的關係(描述性統計)-----------------------------------60
表2-6-2 匹茲堡睡眠品質量表效率(efficiency)在不同基因型與診斷的關係(推論性統計)-----------------------------------60
表2-7-1 匹茲堡睡眠品質量表困擾(disturbances)在不同基因型與診斷的關係(描述性統計)---------------------------------61
表2-7-2 匹茲堡睡眠品質量表困擾(disturbances)在不同基因型與診斷的關係(推論性統計)---------------------------------61
表2-8-1 匹茲堡睡眠品質量表用藥(medications)在不同基因型與診斷的關(描述性統計)------------------------------------62
表2-8-2 匹茲堡睡眠品質量表用藥(medications)在不同基因型與診斷的關(推論性統計)------------------------------------62
表2-9-1 匹茲堡睡眠品質量表白天功能(dysfunction)在不同基因型與診斷的 關係(描述性統計)------------------------------63
表2-9-2 匹茲堡睡眠品質量表潛伏期(latency)在不同基因型與診斷的關係(推論性統計)-------------------------------------63
表3-1 MTERFD3(rs2287161)SNP不同基因型對自殺的發生沒有影響合併病人組及健康組分析統計------------------------------64
表3-2 MTERFD3(rs2287161)SNP不同基因型對自殺的發生沒有影響病人組、健康組分析統計---------------------------------65
表4-1 MTERFD3(rs2287161)SNP不同基因型對焦慮的發生沒有影響合併病人組及健康組分析統計表----------------------------66
表4-2 MTERFD3(rs2287161)SNP不同基因型對焦慮的發生沒有影響病組、健康組分析統計表--------------------------------67
表5-1 MTERFD3(rs2287161)SNP不同基因型對憂鬱的發生沒有影響合併病人組及健康組分析統計表----------------------------68
表5-2 MTERFD3(rs2287161)SNP不同基因型對憂鬱的發生沒有影響病人組、健康組分析統計表-------------------------------69
圖一、 rs 2287161 SNP會影響MTERFD3 啟動子-----------------------------------------------------------------70
附錄
附錄一、MTERF家族蛋白結構----------------------------------------------------------------------------------71
附錄二、MTERFD3基因結構與rs 2287161 SNP位點相對位置----------------------------------------------------------72
附錄三、選殖不同SNP的定序結果-------------------------------------------------------------------------------73
附錄四、探討MTERFD3 (rs2287161) SNP變異位C和G 對該基因啟動子活性研究流程---------------------------------------74
附錄五、PSQI睡眠品質問卷-----------------------------------------------------------------------------------76
附錄六、自殺、焦慮、憂鬱問卷題目-----------------------------------------------------------------------------77
附錄七、IRB同意臨床試驗證明書-------------------------------------------------------------------------------78
附錄八、國立成功大學醫學院附設醫院臨床試驗說明及同意書-----------------------------------------------------------79
附錄九、國立成功大學基因相關研究同意書-------------------------------------------------------------------------83
附錄十、收案流程-------------------------------------------------------------------------------------------88[[abstract]]中文摘要
背景：
在一般人群中很常見的情感性疾患，主要是重度憂鬱症(MDD)和雙極性情感性障礙疾病(BPD躁鬱症，包括BP I第一型躁鬱症和BP II第二型躁鬱症)。然而，很少有人知道情感性疾患和基因之間的臨床特徵。本研究目的是探討情感性疾患與相互關聯的MTERFD3基因單一核苷酸多型性之間的臨床特徵。
材料與方法：
這項研究中有1242位參加者，464位個案，522位家屬和256位健康控制組，2008年至2012年在台灣南部及北部共六個臨床單位收案，包括診所、區域醫院或醫學中心。參加者接受訓練過的研究助理，採用複合性國際診斷訪談問卷(CIDI)，收集人口學和臨床特徵的資料。資料的收集來自重度憂鬱或躁鬱症患者，以及他們的家屬和其他無血緣健康成員。我們探討MTERFD3基因單一核苷酸多型性與臨床特徵間的相關性研究，也轉殖MTERFD3基因的啟動子，觀察單一核苷酸多型性對啟動子活性的影響。
結果：
使用PSQI匹茲堡睡眠品質量表，評估在過去的一個月受試者的睡眠情況，在個案與家屬和控制組間有顯著差異。該項目含睡眠品質 (p<0.001)、潛伏期 (P <0.001)、效率 (P <0.001)、睡眠困擾(P <0.001)、使用安眠藥 (P <0.001) 和白天功能 (P <0.001)。但是與睡眠時間沒有顯著差異(P =0.274)。接下來分別分析量表總分和七項指標在不同基因型間與診斷之間的關係，分別比較三基因型(CC、CG、GG)或合併C基因型(CC／CG、GG)或G基因型(CC、CG／GG)進行兩組比較。整體言之，不同診斷之間常有顯著性差異，除品質指標在CC／CG與GG比較時兩因子有交互作用外，其餘皆無交互作用。而單一核苷酸多型性不同基因型對自殺、焦慮與憂鬱的發生也沒有影響。為進一步分析該單一核苷酸多型性是否對特定族群的影響較大，分別探討CC、CG、GG三基因型或合併CC／CG型與GG型比較或合併CG／GG型與CC型比較，以及將病人組和健康組合併單獨進行比對分析，結果發現自殺、焦慮、憂鬱與躁症和該單一核苷酸多型性均沒有顯著相關。但在探討單一核苷酸多型性對基因啟動子活性的影響上，我們發現rs2287161單一核苷酸多型性會影響MTERFD3啟動子活性，G較C的啟動子活性為高。
結論：
從這個研究結果可以為我們提供更多的診斷資訊和醫療資源利用，做為未來預防精神疾病發展的參考。
Abstract
Backgrounds:
Affective disorders, mainly depressive (MDD) and bipolar disorders (BPD, including BP I and BP II), are common in the general population. However, little is known between genes and their clinical features. The aim of this study is to correlate SNP rs2287161 of MTERFD3 gene with clinical features.
Materials and Methods:
There were 1242 participants in this study, including 464 probands, 522 relatives and 256 controls from six clinics, regional hospitals or medical centers in the southern and northern Taiwan from 2008 to 2012. Participants were interviewed by well-trained interviewers using Composite International Diagnostic Interview (CIDI) to collect data on demographic and clinical features. The data were collected from patients with depression or bipolar disorder, their families and other normal people. We also cloned the promoters with different SNPs of MTERFD3 gene to study the correlation of SNPs and clinical features. The promoter activities were measured by Dual luciferase assay system and in a luminometer.
Results:
The Pittburgh Sleep Quality Index was used to assess the sleep situation of subjects in the past month. There were significant differences among probands, relatives and controls. The items are quality (p<0.001), latency (p<0.001), efficiency (p<0.001), disturbances (p<0.001), use of medications (p<0.001) and day-life dysfunction (p<0.001). However, there was no significant difference in sleep duration (p=0.274). To study the interaction of diagnoses (BPI and II) and the SNP rs2287161 of MTERFD3 in PSQI, we didvided the all cases into three genotype groups (CC, CG and GG) or two groups (CC/CG, GG or CG/GG, CC). In general, the scores of PSQI are signicantly different in disgnosis. Except for quality in comparison of groups CC/CG and GG, there are no interactions of diagnosis and this SNP of MTERFD3in other items. We also correlated the different behaviors, such as suicide, anxiety and depression, with this SNP of MTERFD3 and found that this SNP is not associated with these behaviors. To further study the correlation of behaviors and this SNP, we analyzed the data of controls and patients individually and found that this SNP is not associated with these behaviors. To understand if this SNP inloves in MTERFD3gene expression, we cloned the promoter fragments with different alleles on this SNP and found that G allele exerts higher promoter activity than C.
Conclusion:
The results from this study may provide us more information for development of diagnosis and medical resource utilization for preventing mental diseases in the future
Study of the inhibitory effect and mechanism of chalcone on human bladder cancer cells invasion and migration
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中文摘要……………………………………………………………………...........i
Abstract……………………………………………..…………………………….iii
縮寫表……………………………………………………………………………..v
致謝………………………………………………………………………………vii
目錄……………………………………………………………………………….ix
圖表目錄………………………………………………………………………….xi
第一章 文獻資料回顧
第一節 膀胱癌………………………………………………………………..-1-
第二節 癌細胞的入侵與轉移………………………………………………..-4-
第三節 癌細胞入侵與轉移之相關因子……………………………………..-6-
第四節 細胞外基質的組成與分類…………………………………………-15-
第五節 Chalcone的背景介紹……………………………………...……….-16-
第二章 研究動機……………………………………………………………...-18-
第三章 研究架構……………………………………………………………...-19-
第四章 實驗材料及方法
第一節 實驗材料及實驗設備………………………………………………-20-
第二節 實驗方法……………………………………………………………-27-
第五章 實驗結果…………………………………………………………...…-35-
第六章 討論……………………………………………………………….…..-40-
第七章 結論……………………………………………………………….…..-45-
第八章 參考文獻………………………………………………………….…..-46-
第九章 圖表與圖表說明…………………………………………………...…-63-
第十章 附圖表………………………………………………………………. -79-[[abstract]]Chalcone 是由明日葉所萃取出來的類黃酮抗氧化物，具有抗菌、抗發炎的功效，近來有研究指出 chalcone 已被應用於抗癌的研究。本篇研究主要探討 chalcone 是否可以有效地抑制人類膀胱癌細胞的轉移及侵襲能力，進一步探討相關的分子作用機制。在體外實驗中，為了要觀察 chalcone對於癌細胞轉移能力的影響。我們選取了一株俱有高度轉移能力的人類膀胱癌 T24 細胞，分別處理不同濃度的 chalcone 來探討此藥物對於癌細胞移動性及侵入能的影響。首先利用 wound healing assay 及 Boyden chamber assay ，我們發現chalcone 可以抑制 T24 膀胱癌細胞的移動性及侵入性，在 gelatin zymography、casein-plasminogen zymography 和 polymerase chain reaction 中也發現 chalcone 可以抑制 T24 細胞中MMP-2、MMP-9 及 u-PA 的活性及
messenger RNA 的表現。為了進一步探求 chalcone 抑制 T24 細胞侵入的機轉，利用西方墨點法，我們發現訊息傳遞途徑中的FAK、ERK1/2、p38、JNK1/2 和 Akt 的磷酸化表現皆會受到 chalcone 所抑制。然而這些蛋白的活化是否調控著 T24 細胞的侵襲能力，透過專一性抑制劑：U0126、SB203580、SP600125 及 LY294002 幫助我們釐清了 ERK1/2、p38、JNK1/2 及 PI3K 路
徑可以調控 T24 細胞 MMP-2、MMP-9 及u-PA 的表現。另外在實驗中也證實 small GTPase family，如：Ras、Rac-1、Cdc42、RhoA、RhoB 的蛋白表現量也會受到 chalcone 所抑制。此外，核蛋白中 NF-κB、c-Fos、c-Jun 的表現皆會受到 chalcone 所抑制。綜合上述所有的實驗結果，chalcone 可能是透過FAK/MAPK 和 FAK/PI3K/Akt signal pathway 來影響下游的轉錄因子
NF-κB 的活性，進而影響分泌 MMP-2 、 MMP-9 及 u-PA，最後抑制 T24 細胞的侵襲及移動。
Chalcone is a flavonoid antioxidant extracted from Angelica keiskei koidzumi. It has various effect such as antibacterial and anti-inflammatory activities. In recent studies, chalcone has shown its anti-cancer property. The purpose of this study is to investigate the inhibitory effect and mechanism of
chalcone on human bladder cancer cells metastasis in vitro. We chosed a highly metastastic human bladder T24 cancer cell line which was treated with various concentrations of chalcone to investigate the potential of inhibiting cancer cell migration and invasion. First, we found that chalcone inhibited T24 cells migration and invasion via wound healing assay and Boyden chamber assay. MMP-2, MMP-9, u-PA activity and messenger RNA level were also inhibited by chalcone via gelatin zymography, casein-plasminogen zymography and polymerase chain reaction. In the signal transduction pathway, we found that the phosphorylation of the FAK, ERK1/2, p38, JNK1/2 and Akt were inhibited by chalcone via Western blotting. To verify the activation of above proteins whether regulated T24 metastatic ability, we used specific inhibitors：U0126, SB203580, SP600125 and LY294002 to clarified that the signal transduction of FAK, ERK1/2, p38, JNK1/2 and PI3K/Akt regulated MMP-2 , MMP-9 and u-PA expression. On the other hand, chalcone inhibited the protein level of Ras, Rac-1, Cdc42, RhoA and RhoB with concurrent reduction in the expression level of NF-κB, c-Fos and c-Jun. In the present study, we demonstrated that chalcone inhibited T24 cells might be through the FAK/MAPK and FAK/PI3K/Akt signal pathways and exerted inhibitory effect on the expression of NF-κB transcription factor, MMP-2, MMP-9 and u-PA, afterward inhibited migration and invasion in T24 human bladder cancer cell. This study suggests a possible role of chalcone in bladder cancer therapy
A study on the relation of life stress, social support and happiness in the students of university of medical technology
No full text論文口試委員審定書 i
授權書 ii
誌謝 iii
摘要 iv
Abstract v
目 錄 vi
表目錄 viii
圖目錄 xi
第一章 緒論 1
第一節 研究動機與目的 1
第二節 研究問題 3
第三節 名詞解釋 3
第四節 研究範圍與限制 5
第五節 研究流程 6
第二章 文獻探討 8
第一節 科技大學的相關研究 8
第二節 生活壓力的相關研究 9
第三節 社會支持的相關研究 12
第四節 幸福感的相關研究 15
第三章 研究方法 17
第一節 研究架構 17
第二節 研究假設 18
第三節 研究對象 18
第四節 資料處理 19
第五節 研究工具 21
第四章 結果與討論 34
第一節 不同背景變項分布情形 34
第二節 生活壓力、社會支持與幸福感之現況 38
第三節 不同背景學生對生活壓力、社會支持與幸福感之差異 45
第四節 生活壓力、社會支持與幸福感之相關情形 77
第五章 結論與建議 80
第一節 結論 80
第二節 建議 84
參考文獻 85[[abstract]]本研究主要目的為瞭解科技大學學生的生生活壓力、社會支持與幸福感之現況，與在不同不同背景變項情況下的差異情形，並瞭解三個變項之間的關係。
本研究採問卷調查法，問卷主要分成生活壓力、社會支持、幸福感三個部份，以某醫事科技大學的成年學生為調查對象，結果使用SPSS17.0統計軟體進行分析，來取得本研究所需資料。結果發現如下：
一、學生的生活壓力為中等程度，且會因為性別、家庭狀況、居住狀況、打工狀況的不同而有顯著差異。
二、學生的社會支持為中等程度，且會因為性別、家庭狀況、有無兄弟姊妹、打工狀況的不同而有顯著差異。
三、學生的幸福感為中等程度，且會因為性別、學制、家庭狀況、打工狀況的不同而有顯著差異。
四、生活壓力與社會支持的關係為高度負相關；社會支持與幸福感的關係為高度正相關；生活壓力與幸福感的關係為高度負相關。
依據本研究的結果，建議維持雙親家庭的和諧，有助於增加學生的幸福感；保持身體健康狀況良好，亦顯著影響學生的幸福感。
The aim of this investigation is to understand the state of life stress, social support, and happiness from students of university of technology, as well as the difference between the different demographics, and to understand the relationship between the three variables.
The investigation uses questionnaires. The questionnaires are split into three parts – life stress, social support and happiness. Students from the University of Medical Technology were the participants. Using SPSS17.0 software with analysis to obtain the information needed. The result is shown as below:
1) Students’ life stress, social support and happiness are at a moderate level.
2) The difference in life stress is due to gender, family situation, living situation, and employment or unemployment.
3) The difference in social support is due to gender, family situation, siblings, and employment or unemployment.
4) The difference in happiness is due to gender, education, family situation, and employment or unemployment.
5) Life stress and social support are negatively correlated
6) Social support and happiness are positively correlated
7) Life stress and happiness are negatively correlated
The results from this investigation, some suggestions were made for the University students to refer to
The Effects of Selenium Compounds on Proteomic Expression and Oxidative Stress in Aged Mice and Rat Testis.
No full text目錄........................................................................................................... ii
圖目錄 .................................................................................................... vi
表目錄 .................................................................................................. viii
縮寫對照表 ...................................................................................... 9
中文摘要.............................................................................................. 11
Abstract ............................................................................................... 13
第一章 緒論 ...................................................................................... 15
第一節 前言 .............................................................................. 15
第二節 文獻回顧 ................................................................16
一、 老化對生理狀況之影響……………………...…… 16
1. 老化對生理狀況之影響……………...……...… 16
2. 老化對睪丸之影響.............................................. 16
二、 老化對抗氧化防禦系統之影響 ................................ 17
1. 抗氧化防禦系統與老化之影響 ....................... 18
三、 硒之介紹 .................................................................. 21
1. 硒之介紹 ........................................................... 21
2. 硒之缺乏症及硒中毒 ................................................... 22
3. 硒之化學形式 ................................................... 23
4. 硒的消化、吸收及代謝 ................................... 25
5. 硒之功效 ........................................................... 27
6. 硒對睪丸之影響................................................. 28
四、 老化對睪丸蛋白質體之影響 .................................. 28
1. 蛋白質體學 (proteomics) 之定義 .................. 28
2. 蛋白質體學之研究工具 ................................... 29
3. 蛋白質體學在睪丸組織之研究 ....................... 30
第三節 研究目的與設計 .......................................................... 32
一、 研究目的 .................................................................. 32
二、 研究設計 .................................................................. 32
三、 實驗設計說明 ........................................................... 34
第二章 材料與方法 .......................................................................... 36
一、 動物飼養 .................................................................. 36
二、 飼料之製備 .............................................................. 37
三、 動物犧牲 .................................................................. 40
四、 血樣收集 .................................................................. 40
五、 組織均質 ................................................................. 41
六、 蛋白質測定 ............................................................... 41
七、 免疫轉印法 ......................................................... 43
1. SDS膠體電泳 ................................................... 43
2. 蛋白質免疫轉印法 .......................................... 47
3. 酵素免疫染色法 ............................................... 49
4. 膠體染色 ........................................................... 50
5. 膠體乾片製作 ................................................... 51
八、 蛋白質體學分析 ....................................................... 52
1.睪丸均質液製備............................................. 52
2.睪丸組織透析及蛋白質萃取與收集 ............... 53
3. 蛋白質回溶 ...................................................... 54
4. 蛋白質測定 (2-D Quant kit) ........................... 54
5. 覆水作用 (rehydration) .................................... 55
6. 第一維：等電點聚焦 (IEF) ............................ 56
7. 平衡作用 (equilibration) .................................. 57
8. 第二維：SDS膠體電泳 ................................... 58
9. 膠體染色 .......................................................... 60
10. 膠體影像處理與比對 ..................................... 62
11. 膠片保存 ........................................................ 62
12. 膠內酵素消化.................................................... 63
13. 蛋白質身分鑑定................................................ 64
九、 血漿中ORAC總抗氧化能力測定.......................... 64
十、 組織中TBARS含量之測定 ................................ 65
十一、 組織中Catalase活性之測定 ................................ 67
十二、 組織中GPx活性之測定 ...................................... 68
十三、 組織中SOD活性之測定 ..................................... 69
十四、 組織中維生素C含量之測定 .............................. 70
十五、 組織中GSH含量之測定......................................... 71
十六、 組織中γ-GTP活性之測定...................................... 73
十七、 統計 ................................................................... 74
第三章 硒補充對老齡BALB/c小鼠睪丸氧化壓力及蛋白質體變化之影響
結果...................................................................................... 75
一、BALB/c male mice生長與攝食情形 ............................... 75
二、BALB/c male mice組織重量 ........................................... 75
三、硒補充對小鼠血漿中總抗氧化能力之影響.................... 75
四、硒補充對小鼠睪丸中脂質過氧化程度之影響................ 76
五、硒補充對小鼠睪丸中酵素性抗氧化系統之影響............. 76
六、硒補充對小鼠睪丸蛋白質體之影響.................................... 77
七、硒補充對老齡小鼠GRP78蛋白質表現量之影響..........77
第四章 硒補充對老齡SD大鼠睪丸氧化壓力、GRP78蛋白質表現及
γ-GTP活性之影響 ................................................................... 92
一、SD male rat 生長情形 ............................................. 92
二、SD male rat 睪丸組織重量 ........................................... 92
三、硒補充對大鼠睪丸中脂質過氧化程度之影響................ 92
四、硒補充對大鼠睪丸中酵素性抗氧化系統之影響................ 93
五、硒補充對老齡大鼠GRP78蛋白質表現量之影響…….....94..
六、硒補充對老齡大鼠睪丸中γ-GTP活性之影響................ 94
第五章 討論 ..................................................................................... 93
一、硒補充延緩小鼠老化之過程............................................ 105
1. 硒補充對老齡小鼠體內氧化壓力之影響 ............... 105
2. 硒補充對老齡小鼠體內抗氧化防禦系統之作用... 105
3. 硒補充對小鼠睪丸蛋白質體影響之討論..................... 107
4. 硒補充對小鼠睪丸中GRP78蛋白質表現之影響….... 113
二、硒補充延緩大鼠老化之過程.............................................. 114
1. 硒補充對老齡大鼠體內氧化壓力之影響 .................114
2. 硒補充對老齡大鼠體內抗氧化防禦系統之作用…..….114
3. 硒補充對大鼠睪丸中GRP78蛋白質表現之影……......117
4. 硒補充對大鼠睪丸中γ-Glutamyl Transpeptidase活性之影響...................................................................... 117
第五章 綜合討論與未來展望..........................................................119
參考文獻 ...........................................................................................120
圖目錄
圖1-1 活性氧物質ROS之生成與代謝……………………………..............18
圖1-2 抗氧化防禦系統之作用機制 ...............................................19
圖1-3 GSH反應機制 ........................................................................20
圖1-4 硒之代謝路徑 ........................................................................27
圖1-5 基因體和蛋白質體的生化相關性 ........................................29
圖1-6 蛋白質體學研究的基本方法 ................................................31
圖1-7 動物實驗架構(BALB/c male mice)............................................33
圖1-8 動物實驗架構(SD male rat)............................................ 34
圖2-1 GSH 測定原理圖 ....................................................................72
圖2-2 γ-glutamyl transferase原理…………………......................73
圖3-1 餵食老齡小鼠組(OC、SE與SY)及年輕組(YC)三種試驗飼料七週後 之小鼠血漿中抗氧化能力之比較 ................................................ 81
圖3-2 餵食老齡小鼠組(OC、SE與SY)及年輕組(YC)三種試驗飼料七週後 之小鼠睪丸中TBARS活性之影響....................................................82
圖3-3 餵食老齡小鼠組(OC、SE與SY)及年輕組(YC)三種試驗飼料七週後 之小鼠睪丸中catalase活性之影響...................................................83
圖3-4 餵食老齡小鼠組(OC、SE與SY)及年輕組(YC)三種試驗飼料七週後之小鼠睪丸中GPx活性之影響........................................................ 84
圖3-5 餵食老齡小鼠組(OC、SE與SY)及年輕組(YC)三種試驗飼料七週後之小鼠睪丸中SOD活性之影響..........................................85
圖3-6 餵食試驗飼料七週後，老齡控制組(OC)與年輕控制組(YC)小鼠睪 丸二維電泳圖譜之比較 ................................................86
圖3-7 餵食試驗飼料七週後，老齡控制組(OC)與無機硒補充組(SE)小鼠睪 丸二維電泳圖譜之比較 ........................................................87
圖3-8 餵食試驗飼料七週後，老齡控制組(OC)與有機硒補充組(SY)小鼠
睪丸二維電泳圖譜之比較 ..............................................88
圖3-9餵食老齡小鼠組(OC、SE與SY)及年輕組(YC)三種試驗飼料七週後之小鼠睪丸中GRP78表現量之影響............................................91
圖4-1 餵食老齡大鼠組(OC、LSE、HSE、LSY與HSY)及年輕組(YC)五種試驗飼料十二週後之大鼠睪丸中TBARS活性之影響....................97
圖4-2 餵食老齡大鼠組(OC、LSE、HSE、LSY與HSY)及年輕組(YC)五種 試驗飼料十二週後之大鼠睪丸中catalase活性之影響....................98
圖4-3 餵食老齡大鼠組(OC、LSE、HSE、LSY與HSY)及年輕組(YC)五種試驗飼料十二週後之大鼠睪丸中GPx活性之影響........................99
圖4-4 餵食老齡大鼠組(OC、LSE、HSE、LSY與HSY)及年輕組(YC)五種
試驗飼料十二週後之大鼠睪丸中SOD活性之影響.............100
圖4-5 餵食老齡大鼠組(OC、LSE、HSE、LSY與HSY)及年輕組(YC)五種試驗飼料十二週後之大鼠睪丸中維生素C含量之影響............101
圖4-6 餵食老齡大鼠組(OC、LSE、HSE、LSY與HSY)及年輕組(YC)五種
試驗飼料十二週後之大鼠睪丸中GSH含量之影響…...........102
圖4-7餵食老齡大鼠組(OC、LSE、HSE、LSY與HSY)及年輕組(YC)五種試驗飼料十二週後之大鼠睪丸中GRP78表現量之影響.......................................................................................................103
圖4-8餵食老齡大鼠組(OC、LSE、HSE、LSY與HSY)及年輕組(YC)五種試驗飼料十二週後之小鼠睪丸中r-GTP活性之影響..............104
表目錄
表5-1 硒補充對小鼠睪丸組織氧化程度與抗氧化系統之影響表1-1 已知哺乳動物體內硒蛋白質其主要功能........................................20
表1-2 無機硒、有機硒之化學結構 .............................................24
表1-3 硒蛋白 (selenoproteins) ......................................................25
表2-1 BALB/C試驗飼料之組成 .............................................................38
表2-2 SD male rat試驗飼料之組成 ........................................................39
表3-1 餵食老齡小鼠組 (OC、SE與SY) 及年輕組 (YC) 三種試驗飼料七 週後小鼠最終體重、體重增加量、飼料增加量及飼料利用效率....................................................................................................79
表3-2 餵食老齡小鼠組 (OC、SE與SY) 及年輕組 (YC) 三種試驗飼料七 週後小鼠睪丸組織重量與相對重量..........................................80
表3-3 餵食試驗飼料七週後，小鼠睪丸二維電泳有差異蛋白質點之比較 ....................................................................................................89
表3-4 餵食試驗飼料七週後，小鼠睪丸二維電泳有差異蛋白質點之功能 ...................................................................................................90
表4-1 餵食老齡小鼠組 (OC、LSE、HSE、LSY與HSY)及年輕組 (YC) 五
種試驗飼料十二週後小鼠最終體重、睪丸組織重量及相對重
量....................................................................................................96
表5-1 硒補充對小鼠睪丸組織氧化程度與抗氧化系統之影響………106
表5-2 硒補充對大鼠睪丸組織氧化程度與抗氧化系統之影響……....116[[abstract]]隨著年齡的增長，睪丸功能如製造精子及相關荷爾蒙作用減少，其原因與睪丸氧化傷害增加有關。在睪丸中含有高量的硒，許多研究顯示硒具抗發炎、抗腫瘤等作用，其作用機轉與抗氧化能力有關。在老化過程中，氧化傷害的累積，是導致老化相關疾病主要原因。本研究目的在探討兩種形式硒補充對老齡鼠睪丸氧化狀態及蛋白質體之影響。以sodium selenite作為無機硒來源，以selenium yeast作為有機硒的來源，採用64週BALB/c雄性小鼠，依體重隨機分成三組，分別餵食不同實驗飼料：控制組(OC)、無機硒組(SE；0.5 mg/kg diet)、有機硒組(SY；0.5 mg/kg diet)，另以6週齡之年輕鼠作為年輕控制組(YC)，餵食7週，分析各組睪丸組織液均質液之抗氧化酵素活性，發現OC組氧化壓力明顯高於YC組，補充硒可以改善氧化壓力的增加。另以二維式電泳比較各組睪丸蛋白質體的變化，結果發現了有7個蛋白質點為OC組明顯高於YC組，且SE組與SY組明顯較OC組為低，經質譜儀分析鑑定鑑定其蛋白質身分,其中包含有3-hydroxyisobutyrate dehydrogenase；protein disulfide-isomerase A3；78 kDa glucose-regulated protein(GRP78)等等。顯示這些蛋白質點與老化有關，且可經由補充硒而減少其因老化而增加。之後將78 kDa glucose-regulated protein (GRP78)做進一步western blot分析，發現睪丸中的GRP78表現量OC組較YC組低，而在補充不同型式的硒後，GRP78表現量則有明顯的上升。另外採用79週SD雄性大鼠，依體重隨機分成五組，分別餵食不同實驗飼料：控制組(OC)、低劑量無機硒組(LSE；0.5 mg/kg diet)、高劑量無機硒組(HSE；2 mg/kg diet)、低劑量有機硒組(LSY；0.5 mg/kg diet)、高劑量有機硒組(HSY；2 mg/kg diet) ，另以14週齡之年輕鼠作為年輕控制組(YC)，餵食12週，分析各組睪丸組織液均質液之抗氧化酵素活性，以及western blot分析其睪丸組織中GRP78表現量，另測定其γ-GTP活性，發現大鼠之結果與小鼠睪丸分析之結果相比，有相同的趨勢。本研究顯示，給予硒補充可改善老齡小鼠睪丸抗氧化能力，且影響睪丸蛋白質的表現，此結果可以作為預防睪丸功能退化之參考。
Increase with age, testicular function, such as the manufacture of sperm and hormonal effects reduce the increase in its causes and testicular oxidative damage. Contain high levels of selenium in the testis, many studies have shown that selenium anti-inflammatory, anti-tumor effects, its mechanism of action and antioxidant capacity. The purpose of this study was to investigate the two forms of selenium supplement on aging mice testis oxidation state and proteomics. 64 weeks of BALB / c male mice were randomly divided into three groups according to body weight, and were fed to experience feed: control group (OC), inorganic selenium group (SE; 0.5 mg/kg diet), and organic selenium group (SY; 0.5 mg/kg diet). We use 6-week-old young rats, as the young control group (YC). Seven weeks after feeding, analysis of testicular tissue fluid homogenate antioxidant enzyme activity was found in the oxidative stress of the OC group which was significantly higher than the YC group; therefore, we found that selenium supplementation can improve the increased oxidative stress. In the testis protein changes of two-dimensional gel electrophoresis, we found seven protein spots for the OC group was notably higher than the YC group, and the SE group with SY group was obviously lower than the OC group identified by identification by mass spectrometry identity of their protein, which contains 78 kDa glucose-regulated protein (GRP78). These proteins are associated with aging; through selenium supplementation they can reduce the increase due to aging. Then we analyze 78 kDa glucose-regulated protein (GRP78) with western blot for further and discover that the expression of GRP78 in the OC group testicles is lower than the YC group, while the complement of different types of selenium, the performance showed a significant increase. Additionally, we adopt 79-week-old SD male rats randomly divided into five groups according to body weight, fed with experience feed: control group (OC), inorganic selenium group (LSE; 0.5 mg/kg diet、HSE; 2 mg/kg diet), and organic selenium group (LSY; 0.5 mg/kg diet、HSY; 2 mg/kg diet). At the other side, 14-week-old young rats, as the young control group (YC). Twelve weeks after feeding, analysis of testicular tissue fluid homogenate antioxidant enzyme activity was found in the oxidative stress, western blot analysis of GRP78 expression in the testes. The γ-GTP activity was measured; we found the rats of the results compared to the results of the analysis of the mouse testis, their tendencies are the same. This study shows that, given selenium supplements can improve the antioxidant capacity of the testes of aging, and affect the performance of testicular protein. This result can be used as the reference to prevent the degradation of testicular function
The Role of IL-7 in Fibrosis Regulation of Renal Proximal Tubular Epithelial cells
No full text口試委員會審定書…………………………………………………i
授權書…………………………………………………………...….ii
誌謝………………………………………………………………....iii
中文摘要……………………………………………...……………iv
英文摘要………………………………………………………...…v
縮寫表…………………………………………………………....…vi
第一章 緒論 1
一、糖尿病腎病變之盛行率 2
二、糖尿病腎臟纖維化之致病與機轉 3
三、腎臟纖維化與EMT過程之相關 6
四、高糖與糖尿病腎病變之關係 8
五、乙型轉型生長因子在糖尿病腎病變中扮演之角色………10
六、纖維化之訊息傳導路徑 ...13
七、白介素-7簡介 18
第二章 研究目的 22
第三章 材料與方法 24
一、試劑 25
二、方法 25
1. 細胞培養 26
( 1 ) 繼代細胞 26
( 2 ) 活化細胞 26
( 3 ) 細胞保存 27
2. 細胞數目 27
3. 酵素連結免疫吸附分析 ( ELISA ) 27
4. 免疫螢光分析 ( Immunofluorescence ) 28
5. 細胞蛋白質總量測定 28
6. 西方墨點分析 29
7. 統計分析 30
第四章 結果 31
一、IL-7對27.5 mM的D-glucose誘導纖維化情形下腎近端小管細胞生長的影響 ….. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...…32
二、IL-7對27.5 mM的D-glucose誘導纖維化情形下腎近端小管纖維蛋白的影響……………………………………………………32
三、IL-7減少27.5 mM的D-glucose誘導腎近端小管細胞外分泌TGF-β1.. .….….….….….….….….….….….….…..….….….…33
四、IL-7對27.5 mM的D-glucose誘導纖維化情形下腎近端小管細胞TGF-β1受器的影響……………………………………………34
五、IL-7對27.5 mM的D-glucose誘導下腎近端小管細胞Smad2/3, pSmad2/3, Smad4 以及 Smad7的影響………………...………34
六、IL-7抑制27.5 mM的D-glucose誘導下腎近端小管細胞胞內上皮型態的影響……………………………………………...…….35
七、IL-7對27.5 mM的D-glucose誘導下腎近端小管細胞於免疫螢光觀察上皮型態的影響……………………………………..…..36
八、IL-7抑制27.5 mM的D-glucose誘導下腎近端小管細胞纖維化的調控機制…………………………………………………....37
第五章 討論 …….38
第六章 結論 42
第七章 圖表 44
第八章 參考文獻 53[[abstract]]背景：在糖尿病腎病變造成的纖維化中，高血糖是一個重要的危險因子。先前的研究發現，IL-7具有抗肺纖維化的潛力。然而，IL-7在腎纖維化的過程扮演的角色未知。因此，我們想探討IL-7在腎近端小管上皮細胞高糖的環境下所扮演的角色。方法：細胞培養在高糖最終濃度為27.5 mM兩天，在最後24小時加入不同濃度的IL-7 (10、 50、100與 200 ng/ml)。利用ELISA來偵測細胞外分泌的纖維蛋白及乙型生長轉型因子濃度。再使用Western blot來觀察EMT的標記【包括α-smooth muscle actin ( α-SMA) 及 E-cadherin】，訊息傳遞，及EMT的啟動子( Snail, Slug )之表現。以及使用免疫螢光染色來觀察原位蛋白的表現（譬如Fibronectin, α-SMA, E-cadherin, Snail, Slug）。結果：我們發現IL-7抑制高糖誘導減少的細胞生長及抑制高糖誘導的細胞纖維化。並且IL-7能夠阻斷高糖誘導所升高的纖維蛋白，第一型乙型轉型生長因子，第一型乙型轉型生長因子接受器以及磷酸化Smad2/3。相反的，IL-7可以逆轉高糖所誘導減少的Smad7。除此之外，IL-7能夠抑制高糖所誘導增加的α-SMA, Snail及Slug，並且能逆轉高糖誘導減少的E-cadherin。結論：IL-7抑制高糖誘導的腎近端小管纖維化可能通過調節 Smads和EMT的途徑。
Background: Hyperglycemia is the most important risk factor in the progression of renal fibrosis in diabetic kidney. Based on previous studies, interleukin-7 (IL-7) may possess antifibrotic activities in pulmonary fibrosis model. However, the role of IL-7 in the pathogenesis of renal tubulointerstitial fibrosis remains unclear. Thus, we hereby elucidate the effects of IL-7 in cultured renal proximal tubular epithelial cells (designated as HK-2) treated under hyperglycemic media. Material and Methods: Cells were cultured in high glucose (HG, 27.5mM) for 2 days. Different concentration of IL-7 (10、 50、100 or 200ng/ml) was added in the last 24 hours of culture. ELISA was used to evaluate the secreted protein such as fibronectin and TGF-β1. Western blot was used to examine the EMT marker (including α-smooth muscle actin (α-SMA) and E-cadherin), signal transducer (including Smad Smad2/3 and Smad7) and EMT initiator (e.q Snail, Slug). Immunofluorescence staining was used to assay the in situ expression of proteins (e.q. α-SMA, E-cadherin and Snail). Results: We found that IL-7 significantly attenuated HG-inhibited cellular growth and HG-induced fibrosis. HG-induced up-regulation of fibronectin, TGF-β, TGF-β RII and p-smad2/3 was markedly inhibited by IL-7. On the contrary, HG-induced down-regulation of smad7 was significantly reversed by IL-7 instead. Moreover, IL-7 markedly inhibited HG-induced increase in α-smooth muscle actin and snail. Whereas HG-induced decrease in E-cadherin expression was reversed as well. Conclusion, IL-7 has the potential to inhibit high glucose-induced renal tubular fibrosis possibly by modulating Smads and EMT pathway