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Factors Associated with Maternal Choice of Feeding Methods for the Women's of Tainan Area
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Dioscorea alata L. reverses renal interstitial cellular fibrosis by regulating Smad- and epithelial-mesenchymal transition related signaling pathway
Get PDF[[abstract]]腎間質纖維化是糖尿病腎病變重要併發症，其病理特徵包括細胞外基質蛋白累積、纖維母細胞的增生和上皮細胞間質轉換，最後導致末期腎臟疾病。在過去的研究中顯示，乙型轉型生長因子（TGF-β）是一個與細胞增生或上皮細胞間質轉換及腎纖維化相關的致纖維化因子。近年來的文獻發現，山藥為重要糧食和藥用作物，也廣泛應用於臨床上治療糖尿病的中藥材。然而，山藥對腎臟纖維化所調控的機轉及其分子作用機制還不清楚。因此，本研究探討山藥水萃取物對β-羥丁酸所誘導NRK-49F細胞間質纖維化的調控及訊息傳遞過程。我們利用西方墨點法分析TGF-β下游受器（第一型及第二型受器）和Smad訊息蛋白質（如：Smad2/3、pSmad2/3、Smad4和Smad7），以及EMT表現標誌蛋白（α-SMA、MMP-2與E-cadherin）；並以ELISA的方式測定TGF-β的生物活性及fibronectin表現量，再以免疫螢光染色觀察轉錄因子Snail及纖維蛋白fibronectin的表現。實驗結果發現，山藥萃取物顯著地降低β-羥丁酸所誘導Smad蛋白質（如Smad2/3、pSmad2/3、與Smad4）增加，同時也增加β-羥丁酸所減少的Smad7蛋白質。另一方面，山藥顯著地減少間質標記蛋白α-SMA、MMP-2以及增加上皮細胞標記蛋白E-cadherin的表現。上述結果顯示山藥萃取物可降低TGF-β/ smad訊息以及調節上皮-間質轉換，因此，山藥萃取物可做為一種新穎的抗纖維化之標靶藥物。
Renal interstitial fibrosis (RIF) is one of the major complications of diabetic nephropathy and the pathology of RIF consists of accumulation of extracellular matrix protein, proliferation of fibroblasts, and epithelial-to-mesenchymal transition (EMT) that leading to renal fibrosis and end-stage renal disease. Transforming growth factor beta1 (TGF-β1) has been implicated as an inducer of cell proliferation and EMT. It is also known as a potent fibrogenic growth factor in renal fibrosis. Recent studies suggest that yam, an agricultural and medicinal plant, is an important herb in Chinese medicine widely used for the treatment of clinical diabetes mellitus. However, the molecular mechanism of Dioscorea alata (DA) on antagonizing renal interstitial fibrosis has never been fully investigated. Thus, this study aims to investigate the modulatory effect of water extract of DA in NRK-49F cells (a rat fibroblasts cell line) under renal fibrosis model, which was induced by β-hydroxybutyrate (β-HB). Western blotting was used to examine protein expression in TGF-β-related signal proteins (type I and type II TGF-β receptor, Smads2/3, pSmad2/3, Smads4, Smads7) and EMT marker (E-cadherin, α-SMA, MMP-2). In addition, Enzyme-linked immunosorbent assay was used to analyze levels of bioactive TGF-β and fibronectin in the culture media. Expression of Fibronectin and transcription factor Snail, a transcription factor that modulates EMT, was also assessed by immunofluorescence staining. DA extract inhibited β-HB-induced expression of fibronectin in NRK-49F cells (P<0.05) concomitantly with dose-dependent inhibition of Smad2/3, pSmad2/3, Smad4. However, Smad7 was significant increased by DA extract. Mesenchymal marker (α-SMA and MMP-2) was significantly decreased and epithelial marker (E-cadherin) was increased by DA extract. These results suggest that DA extract might down-regulate TGF-β/Smad and modulate EMT. In conclusion, we proposed that DA extract might act as a novel renal fibrosis antagonist for treating diabetic nephropathy
Isolation and Identification of Lactic Acid Bacteria and their Effects on Si–raw Production
No full text[[abstract]]乳酸菌廣泛分布於自然界，其不僅能提升食品風味，同時亦可提供人體生理功能，尤其一些具生理活之性胜肽亦被研究做為機能性成分。因此，本研究嚐試將分離菌株 (Pediococcus acidilactici) 做為菌酛，並接種於肉 (Si-raw)中，測定其在肉中之生長情形、蛋白質分解作用與抗氧化能力。
四株乳酸菌以 MRS培養基自竹筍園的土壤 (TWB002)、新疆酸奶(XIN003) 及泰式酸肉(THA001、THA003) 分離。利用PCR技術分離16S rRNA序列以鑑定細菌。經NCBI BLAST 資料庫比對，鑑定結果顯示TWB002和THA003與P. acidilactici 的Uga146-3及UL5呈現高相似度(98,99%)，而K3和W1則分別與Lactobacillus brevis 的NRIC0134及NRIC0138具高度相似性(99,99%)。
各分離菌株均能於500 ppm 亞硝酸鹽和5000 ppm硝酸鹽濃度下生長。而P. acidilactici TWB002、THA003 及 Lb. brevis XIN003較Lb. brevis THA001能適應更高之食鹽濃度，但隨著食鹽濃度的提高，各菌之生長均受到抑制。較低之pH值亦限制各菌株之生長，尤其pH值降到5.08以下時 (P< 0.05)。然而，較低之pH值和較高之食鹽濃度同時會抑制乳酸菌之酵素活性，因此，P. acidilactici TWB002 呈現出的結果應最適合使用於肉製程中。抗生素敏感性試驗顯示各菌株對抗生素之感受性不同，對Streptomycin、sulphafurazole 、Penicillin-G 及Ampeicillin 均呈現抵抗性，而對Erythromycin則為敏感性。
選擇以P. acidilactici TWB002做為肉的接種菌株，並於25℃培養7天，測定其生長情形及蛋白質分解活性。結果顯示P. TWB002於肉的發酵過程總乳酸菌數維持106-7 cfu/g，產酸能力方面在發酵2天開始會明顯增加 (P<0.05)，但兩組之間並無差異的產生。肌漿蛋白SDS-PAGE膠體電泳中，並未發現處理組與對照組之差異，但可溶性蛋白質比率則會隨發酵時間而減少 ( P<0.05 )，反之胜肽及胺基酸的釋出會隨之增加 ( P<0.05 )。肉發酵期間小分子蛋白質 (M. W. ＜10,000) DPPH和ABTS自由基清除率會隨發酵期間提高 ( P<0.05 )，並且發酵7天分別增加到原來的7.4和20倍 (P<0.05)，發酵5天接種P. acidilactici TWB002之組別，其ABTS之清除率較高於對照組 ( P<0.05 )，此結果顯示其小分子蛋白質可能為生理活性物質，並可做為發展抗氧化機能性成分之來源。
Lactic acid bacteria are widely exist in nature. They were not only improve sensory characteristics of food, but also supply some physiological functions. It was noticed that biologically active peptides have now been studied and designing as functional ingredients. The objective of this study was to evaluate the role of screened natural stain (Pediococcus acidilactici) which was used as a starter in Si-raw, the growth of this organism in Si-raw and their protein breakdown as well as antioxidant ability.
Four lactic acid bacteria were collected from bamboo farm (TWB002)、Xinjang sour milk (XIN003) and Thailand sour pork (THA001、THA003) using MRS agar plate. Three typical lactic acid bacteria colony morphologies were observed as well as strains were selected for further Si-raw studies. To identify the bacteria, we use PCR technique to isolate 16S rRNA. Analysis of the DNA sequences by NCBI BLAST program revealed that TWB002 and THA003 showed highest level of similarity (98, 99% identity) to P. acidilactici strains Uga146-3 and UL5. Where as, XIN003 and THA001 showed highest level of similarity (99, 99% identity) to Lactobacillus brevis strains NRIC0134 and NRIC0138.
Each strain could adapt to 500 ppm nitrite sodium and 5000 ppm of nitrate sodium. P. acidilactici TWB002, THA003 and Lb.brevis XIN003 could also adapt to higher sodium chloride content than Lb. brevis THA001, but less growth was observed when the concentration of sodium chloride increased to higher. The growth of each strains were limited by lower pH value, they did not growth very well until pH drop to 5.08 (P< 0.05). However, lower pH value and higher sodium chloride content also inhibits enzymatic activity of lactic acid bacteria, P. acidilactici TWB002 were the most appropriate to use in the procedure of Si-raw manufacture. The antibiotic sensitivity tests showed that all genus displayed diversity in their ability to resist to the antibiotics. All the strains were resistant to Streptomycin、sulphafurazole 、Penicillin-G, amoxicillin and tetracycline as well as susceptible to Erythromycin. Tetracycline was narrow intermediate the genus of Pediococcus.
P. acidilactici TWB002 was used as a starter culture and inoculated into porcine Si-raw. During fermentation, samples were incubated at 25℃ for 7 days and determined for their growth rates and proteolytic activities. The results showed P. acidilactici grew very well and remained between 5-7 log cfu/g (P<0.05). The result showed that P. acidilactici had acid production activity. The increase in acidity was found when the samples were fermented for 2 days (P<0.05). No remarkable differences were detected between the inoculated sample and control sample on the SDS-PAGE electrophoregram. During fermentation, the ratio of soluble protein decreased with curing time (P<0.05). However, in the presence of Si-raw, proteins were decomposed to small molecules (M. W. ＜10,000) such as peptide and free amino acids. It was found that the ABTS and DPPH scavenging abilities increased with the fermentation time, and the values were about 7.4 and 20 times (P<0.05) after 7 days. Furthermore, higher ABTS radical scavenging abilities were observed from the P. acidilactici inoculated samples than the control samples after fermented for 5 days (P<0.05). These findings suggested that bio-active compounds from the small molecule proteins might be potential resources for the development of antioxidant functions
Study on the effects of organic and inorganic selenium on the anti-inflammation in RAW 264.7 cells and BALB/c aged mice.
No full text目錄 i
圖目錄 vi
表目錄 ix
致謝 x
縮寫對照表 xii
中文摘要 xiii
Abstract xv
第一章 緒論 1
第一節 前言 1
第二節 文獻回顧 3
一、 老化對生理狀況之影響 3
1. 老化指標 3
2. 老化對生物體免疫系統之影響 3
3. 人體抗氧化系統之介紹 6
4. 老化對生物體抗氧化之影響 8
5. 老化對生物體發炎之影響 9
二、 硒之介紹 13
1. 硒之介紹 13
2. 硒之攝取量、缺乏症與硒中毒 14
3. 硒之化學形式 16
4. 硒在生物中之代謝： 18
5. 硒之功效 20
6. 硒之抗發炎作用 22
第三節 研究目的與設計 23
一、 研究目的 23
二、 研究設計 24
三、 實驗設計說明 25
第二章 以LPS誘發一氧化氮生成量探討硒的抗發炎作用 26
第一節 前言 26
第二節 材料與方法 27
一、 細胞株來源及培養條件 27
二、 硒來源 27
三、 細胞存活率分析 27
四、 一氧化氮生成量之分析 29
五、 統計分析 30
第三節 結果 30
一、 不同硒型式補充對RAW 264.7細胞生長之影響 30
二、 不同硒型式補充對NO生成量之影響 30
第四節 討論 36
一、 硒添加對RAW 264.7細胞生長之影響 36
二、 硒添加抑制NO生成量之影響 36
第三章 硒補充對老化發炎作用之影響 38
第一節 前言 38
第二節 材料與方法 39
一、 動物飼養 39
二、 飼料之製備 39
三、 動物犧牲 41
四、 腹腔細胞收集 41
五、 血樣收集 42
六、 一氧化氮生成量之分析 43
七、 PGE2生成量之分析 43
八、 蛋白質測定 45
九、 免疫轉印法 46
1. SDS膠體電泳 46
2. 蛋白質免疫轉印法 51
3. 酵素免疫染色法 53
4. 膠體染色 54
5. 膠體乾片製作 55
十、 組織均質 56
十一、 TBARS測定 56
十二、 ORAC總抗氧化能力測定 58
十三、 組織中單胺氧化酶活性之測定 59
十四、 組織中Catalase活性之測定 60
十五、 組織中GPx活性之測定 61
十六、 組織中SOD活性之測定 62
十七、 組織中維生素C含量之測定 64
十八、 組織中GSH含量之測定 65
十九、 血漿中IFN 活性之測定 66
二十、 血漿中TNF 活性之測定 68
二十一、血漿中IL-6 活性之測定 68
二十二、血漿中IL-1 活性之測定 68
二十三、血漿中IL-2活性之測定 70
二十四、血漿中IL-10活性之測定 70
二十五、尿液中Creatinine含量之測定 70
二十六、尿液中8-iso PGF2活性之測定 71
二十七、尿液中Bicyclo PGE2活性之測定 73
第三節 結果 76
一、 動物生長及攝食情形 76
二、 組織重量 76
三、 硒的補充對小鼠發炎指標之影響 76
四、 硒的補充對小鼠腦部及肝中單胺氧化酶活性之影響 77
五、 硒的補充對小鼠血漿中總抗氧化能力之影響 78
六、 硒的補充對小鼠脂質過氧化指標之影響 78
七、 硒的補充對小鼠抗氧化能力之影響 78
八、 硒的補充對老化小鼠血漿中細胞激素之影響 80
第四節 討論 104
一、 硒補充延緩了老化指標 104
1. 硒補充對動物生長及組織重之影響 104
2. 硒補充對腦部老化及體內氧化壓力改善之影響 105
3. 老化所造成抗氧化系統之改變 107
二、 老化對發炎反應之變化 110
三、 硒減少老化所致之發炎指標 112
四、 有機與無機硒對老齡鼠發炎作用影響之比較 113
第四章 總結及未來展望 116
參考文獻 117
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Tinggi
Development of PCR detection of phytopathogenic bacteria in Phalaenopsis orchids
Get PDF中文摘要
英文摘要
壹、前人研究
一、蘭花簡介
二、蘭花產業的發展狀況
三、蘭花細菌性病害之種類
四、蘭花細菌性病害之DNA檢測法
五、Multiplex PCR
貳、材料與方法
一、供試菌株
二、菌株培養
三、染色體DNA之純化
四、病原細菌核酸序列資料之收集與分析
五、聚合酵素連鎖反應
六、PCR產物之膠體電泳分析
七、PCR產物之純化
八、PCR產物之接合作用
九、大腸桿菌勝任細胞之轉型
十、PCR產物之定序分析
十一、生物資訊分析
十二、專一性測試
十三、靈敏度測試
十四、Multiplex PCR
參、結果
一、褐斑病菌之分析結果
二、葉班病菌之結果分析
三、軟腐病菌之分析結果
四、多引子組之分析結果
肆、討論
一、ITS專一性引子組的開發
二、rpoB及rpoD專一性引子組的開發
三、gyrB專一性引子組的開發
四、idgA及idgB專一性引子組的開發
五、Multiplex PCR檢測技術的開發
六、總結
伍、參考文獻
陸、圖表
參考文獻
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Phalaenopsis became the most important product in the industry of flowers for export, the cultivation quantity of orchids increased rapidly, in recent years. Growing the orchid in the greenhouse caused the increasing of the temperature and humidity and this environment wo
Differences of proteolytic enzymes in permissive and non-permissive hosts for Angiostrongylus cantonensis infection
No full text中文摘要......................... i
英文摘要......................... ii
簡寫表........................... iii
誌謝............................. iv
第一章 緒論..................... 1
第一節 廣東住血線蟲(Angiostrongylus cantonensis) ........1
第二節 血纖維蛋白酶原活化因子(Plasminogen Activator,PAs)..6
第三節 基質金屬蛋白酶(Matrix metalloproteinases, MMPs).. 8
第四節 研究目標........................................ 12
第三章 研究方法........................................ 15
第一節 實驗動物........................................ 15
第二節 幼蟲的製備....................................... 15
第三節 動物的感染....................................... 15
第四節 CSF的細胞計數.................................... 16
第五節 Gelatin and casein substrate zymography........ 16
第六節 統計學的分析..................................... 17
第四章 研究結果........................................ 18
第一節 CSF中嗜伊紅性白血球分析.......................... 18
第二節 CSF與腦中酵素活性分析............................ 18
第五章 討論.............................................19
第六章 結論............................................. 24
參考文獻................................................ 26
附圖................................................... 43
作者簡歷............................................... 46
附圖目錄
Figure 1. Changes in eosinophil counts of the CSF.......43
Figure 2. Enzymatic activity in the CSF.................44
Figure 3. Enzymatic activity in the brain...............45
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The Research Analysis of Janitor Management - to Take Kaishui Vocational Senior High School as an Example
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第一章 緒論 1
第一節 研究背景 1
第二節 研究動機與目的 3
第三節 研究問題、範圍與限制 4
第四節 章節安排與研究流程 5
第五節 名詞解釋 7
第二章 文獻探討 8
第一節 工友管理之演進 8
第二節 工友的角色與功能 10
第三節 工友的薪資與考核 13
第四節 人力運用與管理之理論探討 17
第五節 學校工友管理的相關研究探討 25
第三章 研究設計 30
第一節 研究架構 30
第二節 研究方法 31
第三節 研究對象 36
第四節 研究工具 39
第五節 研究的信度與效度 42
第六節 研究倫理 44
第七節 資料分析步驟 45
第四章 訪談結果分析 48
第一節 工友在學校工作的情況與問題 48
第二節 工友管理的情形 62
第三節 提升工友管理的有效方法 73
第五章 結論與建議 80
第一節 研究發現 80
第二節 建議 83
參考書目 87
中文部分 87
英文部分 90
附錄一 工友管理要點 91
附錄二 事務管理規則 97
附錄三 機關學校工友薪餉核支標準表 103
附錄四 訪談同意書 104
附錄五 訪談內容確認單 105
附錄六 開水工商工友平時考核表 106
附錄七 開水工商工友年終考核表 107
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Weick, K. E.,2001, Making Sense of the Organization. Oxford: Blackwell Publishers.[[abstract]]工友管理之研究分析-以開水工商為例
作者：黃文璉
國立台東大學區域政策與發展研究所公共事務管理在職專班碩士論文
中文摘要
在學校裡，工友的職位最低，待遇最少，且工作內容繁雜，若再加上內外在的種種因素，極易造成管理上的問題。研究者身為工友管理者，責無旁貸的理應重視這群被忽視的默默工作者，因此，本研究藉由文獻分析與深度訪談等研究方法，針對開水工商的工友及其所屬的工作單位主管，藉由質性研究的分析，以了解工友在學校工作的情況與問題，並探討工友管理的情形，進而研擬出提升工友管理的有效方法，希冀提供教育主管機關或相關學校在工友管理工作的實質參考。
經研究分析後發現，工友在工作中所產生的問題如下：
一、工作負荷過大及專業知能不足導致工作壓力大。
二、年齡過大造成學習意願低落。
三、對「工友」的職稱感到自卑，且自我肯定感受偏低。
四、對薪資感到不滿意，且考核的結果亦影響到工友的工作態度。
經研究分析後發現，在工友管理的層面上所產生的問題如下：
一、人力配置不當使得工作與專長無法符合及工作負荷無法均等。
二、工作單位主管對工友的要求不一致，且欠缺工作激勵。
三、工友考核的內容與作法欠缺周延的考量。
經研究分析後發現，研擬出提升工友管理的有效方法如下：
一、建立工友的分工與團體工作制度，且加強工友的學習進修能力。
二、定位工友的角色並提高其自信心，且研議更改適切的「工友」稱謂，以提高社會地位。
三、研議擴大工友薪資級距的範圍或學校輔以工作加班津貼。
四、召開工友座談會，在溝通與了解中調配人力與工作。
五、採取多元溝通的民主領導方式、建立工作單位主管的責任態度。六、擬定公平公正的工友考核制度。
The Research Analysis of Janitor Management - to Take
Kaishui Vocational Senior High School as an Example
Author: Wen-lien Huang
Institute of Regional and Policy Development, National Taitung University
Abstract
Of the bottom rank at the workplace, school janitors are badly paid but given a variety of tasks. Problems of janitor management are likely to emerge if we combine a number of internal and external factors. As a supervisor of janitors at my school, I must take full responsibility for paying more attention to the forgotten group. This paper, therefore, aims to investigate the work situation and problems of school janitors, discuss the ways in which they are managed and suggest effective ways of janitor management by conducting qualitative research analysis and by using the research method of archival research and depth interview. The target group includes janitors and their officers at Kaishui Vocational Senior High School. I hope this report may present itself as a reference for officers at both the Ministry of Education and other schools with similar situations.
After conducting the research analysis, I found that school janitors face work-related problems as follows:
1.pressure of work resulting from a heavy load and a lack of expertise
2.a lack of willingness to learn because of age
3.a sense of inferiority to the title of school janitors and a lack of self- assurance
4.a sense of dissatisfaction with the income; besides, the work attitude of school janitors may be influenced by the results of assessment
After conducting the research analysis, I found the following problems of janitor management:
1.job roles are not properly assigned according to janitors’ expertise; lack of equity in work load
2.the different standards of the officers towards the janitors; lack of incentives
3.the inadequacy of the content and measurements of assessment
After conducting the research analysis, I suggest that the
following ways which might be effective in promoting janitor management:
1.to establish a collaboration and group work system for school janitors and meanwhile enhance their professional development
2.to define the role of school janitors as well as boosting their confidence; to modify the title of janitor so as to elevate their social rank
3.to plan to enlarge the income brackets of school janitors or to reward them with overtime pay
4.to start a meeting in order to understand and communicate with the janitors before the allocation of human power
5.to lead the janitors in a democratic way and by consulting the team; to ask officers of school janitors to build up a sense of responsibility
6.to draw up an equal assessment program for school janitor
The Role of Mst3( Mammalian Ste20 Kinase 3) in Renal Tubulogenesis
No full text研究領域:生物科學類, 生物技術[[abstract]]腎臟發育時，分枝管狀形成的受損，是幼童常見的腎臟衰竭的原因。分枝管狀之形成與細胞之爬行，凋亡及生長週期有關。以HGF 刺激腎臟細胞MDCK，可使其發育成分枝的管狀。當HGF 刺激MDCK，COX-2 蛋白質的表現明顯上升。在缺乏COX-2 的老鼠中，腎臟的分枝過程會受損。另外，腎臟受傷復原時，HGF 接受器，COX-2 以及fibronectin 均會上升。若MDCK 不分泌fibronectin 則管狀分枝無法形成。屬Ste 20 激酶家族的MST3，其蛋白的功能被報導 (1)可促進細胞凋亡 (2) MST3 能控制生長週期 (3) MST3 能抑制細胞移動。這3 項功能均與分化的過程有關。加上我們發現 (i) 在老鼠出生後的腎臟中，MST3 活性上升。(ii) MST3 只有表現在腎臟的細上升枝。(iii) MDCK 大量表現MST3 時， COX-2 明顯上升。 (iv) 必須將細胞培養在舖有fibronectin 之培養皿上，當MDCK 細胞表現大量 MST3 時，才會使細胞移動速度變慢，顯示MST3 與fibronectin 有關。故我們選擇研究MST3 對腎臟發育管狀分枝形成之影響。 In vivo，我們將比較表現活化態MST3 的轉殖性老鼠與野生型MST3 的老鼠，找出MST3 在不同發育時期，在分枝管狀形成的過程，是否有所改變。In vitro，我們將會利用大量表現野生型MST3 及dominant negative MST3 之MDCK 細胞株，研究MST3 是否可經由COX-2 上升，再配合MST3 可增加fibronectin 的分泌而使而促進腎臟分枝發育。再回到in vivo 中，解出MST3 否是經由COX-2 及 fibronectin，控制腎臟分枝管狀發育的詳細機轉。
Disruption of renal branching tubulogenesis during kidney development resulted in renal dysplasia, the major cause of renal failure in young children. The morphogenetic steps are controlled by apoptosis, cell cycle and cell motility. An in vitro model in which HGF induces branching morphogenesis in MDCK epithelial cell culture in three-dimensional collagen gels is established. The COX-2 mRNA and protein expression in MDCK cells are apparently increased after stimulation with HGF. Mouse mutation of COX-2 gene resulted in defects in renal morphogenesis. Besides HGF and COX-2, the interaction of extracelluar matrix (ECM) and cells was also the regulator of branching tubulogenesis. COX-2, HGF receptor, and fibronectin are all increased when mouse was administrated with renal injury. The secretion of fibronectin by MDCK cells promoted renal tubulogenesis. Without the secretion of fibronectin, the tubule could not be formed. The Ste20 family consists of MST1-4 (mammalian Ste20 kinase 1-4)。It was reported that (1) MST3 can promote apoptosis. (2) MST3 can regulate cell cycle and (3) MST3 can inhibit migration. Our results showed (i) the activity of MST3 was elevated in the kidney of 1-2 week old rat (ii) MST3 was exclusively expressed at the thin ascending limb of the kidney. (iii) COX-2 was upregulated in MST3 overexpressed MDCK. (iv) The migration inhibition function of MST3 was appeared when fibronectin was served as the matrix. In vivo, we will compare the active HA-MST3 expressing transgenic mice with wild type MST3 mice to find the effect of MST3 on renal tubulogenesis. In vitro, we will proof that MST3 can stimulate COX-2 to control HGF-induced renal tubulogenesis. Furthermore, HA-MST3 MDCK and dominant negative MST3 will be used to answer if MST3 can secrete fibronectin to lead the formation of renal tubulogenesis. Finally, in vivo and in vitro evidences regarding the relationship between COX-2, fibronectin and MST3 will be considered, rendering new insight into regulating roles for MST3 in renal tubulogenesis
Using Biochip Technology to Elucidate the Anti-Renal Fibrosis Roles and Regulating Mechanism of TGF-Beta/Its Receptors/Smad Related Pathway for Specific Natural Products
No full text研究領域:藥學, 生物技術
計畫編號:NSC96-2314-B273-002-MY3[[abstract]]糖尿病與腎病變是國人十大死因第四名與第八名。據統計，全球有超過200 萬人接受長期洗腎治療，台灣高達四萬人洗腎，慢性腎臟疾病更高達八十萬人，腎病健保支出就高達兩三百億。因糖尿病腎纖維化與腎功能喪失有密切的相關，因此，瞭解腎臟纖維化之分子機轉，並進一步開發出抗腎纖維化之有效方法，便成為當今腎臟學家最重要的課題。本團隊已證明TGF-芻與其兩型受器對於腎纖維化扮演關鍵的角色(三年內於 J Am Soc Nephrol 、Biochem J、J Cell Biochem等國際期刊共有10篇發表)。並已初步證實，天然物中具有活血化瘀之紅花水萃物，可透過壓制TGF-芻路徑而抑制腎纖維母細胞產生纖維。因此，本計畫目標有三，將分別於各年度完成。一、開發抗腎纖維化天然物活性篩選晶片平台，並以紅花等五種活血化瘀中藥進行篩選。二、探討有效成分抗腎纖維作用機轉。三、以活體腎纖維化模式驗證有效成分抗腎纖維之活性。具體作法上，第一年我們利用BMP-2蛋白，一種TGF-芻受器與纖維化抑制蛋白，作為抗腎纖維化的標的分子，並利用液相高效層析配合中草藥晶片，自中草藥萃取物中篩選出可與BMP-2蛋白結合的特性成分，同時解出其化學結構。並利用一系列腎臟細胞株：包含M13(腎絲球細胞)、VEPT (近端腎小管細胞)、MDCK(遠端腎小管細胞)與NRK-49F (腎間質纖維母細胞)，以fibronectin分泌來驗證各成分抗腎細胞纖維化的效應。第二年。我們以西方轉漬搭配RT-PCR與免疫細胞染色等方式，探討各化合物於劑量與時間反應下，對TGF-beta、TGF-beta第一與第二型受器、受器下游訊息傳遞分子（如Smad2,3）與纖維蛋白（fibronectin與IV型collagen），於啟動子活性、mRNA與蛋白質含量表現的影響。第三年，將建立第二型糖尿病腎臟纖維化模型（高果糖餵食大鼠），驗證第兩年所篩選有效成分之抗腎纖維效應。本計畫預期可開發抗腎纖維化天然物活性篩選平台，並透過抗腎纖維活性成分來釐清纖維化作用機轉，預期可提供全新糖尿病腎纖維化之治療方式。
Diabetes and renal disease is the fourth and eighth leading cause of death in Taiwan. According to recent statistics, about two million peoples have to be treated with renal dialysis all over the world. And about 800 thousand Taiwanese peoples are suffered from chronic renal disease. Social cost of renal related diseases from health insurance is higher than two hundred hundred million per year. Since diabetic renal fibrosis is closely related with the loss of renal function, it is important for nephrologists to elucidate the renal fibrogenic mechanism and develop alternative fibrosis antagonizing approaches. Our teams have demonstrated that TGF-芻and its receptors play key roles in renal fibrosis (10 citations published in J Am Soc Nephrol, Biochem J, J Cell Biochem related journals in 3 years). Moreover, we preliminary found that natural product extract from safflower (Carthamus tinctorius L) can suppress fibrosis level in renal fibroblast by suppressing TGF-芻pathway. Thus, the main goals of this 3-year project are: First, a high-throughput herbal chip screening platform for anti-fibrogenic active compounds from 5 herbs with blood-stasis inhibitory activity will be developed. Second, the anti-renal fibrosis mechanism for each compound will be elucidated. Third, a type II diabetes model will be established to verify the renal fibrosis therapeutic efficacy. Procedure: In the first year, BMP-2, a TGF-芻receptor antagonist, will be used as a fibrosis inhibitory marker. HPLC combined with herbochip will be used to establish a screening platform to explore BMP-2 binding fractions (a potential effector on TGF-芻洶signaling) from extract of herbs and to resolve its chemical structure. Moreover, respective renal cell lines including M13 (mesangial cells), VEPT (proximal tubules), MDCK (distal tubules), and NRK-49F (fibroblast) will be used to verify the anti-fibrosis effects of each fractions. In the second year, western blotting, RT-PCR, and immunocytochemistry will be used to explore the time-point and dose-dependent effects of each chemical. Protein/mRNA expression of TGF-芻, both types of TGF-芻receptors, down-stream signals such as Smad2/3, and fibronectin/type IV collagen will be examined as well. In the third year, we will establish a high fructose-fed rat model to validate the efficacy of anti-fibrosis active compounds from the results of first 2 years. Using natural herbs, we do believe having a chance to explore a whole new screening approach and to develop an alternative therapeutic agent for treating renal fibrosis. Moreover, a more clear-cut renal fibrosis-inhibitory mechanism will also be elucidated