Chung Hwa University of Medical Technology
Chung Hwa University of Medical Technology RepositoryNot a member yet
1453 research outputs found
Sort by
Determination of trace elements in herbs at Tainan local market and edible mushrooms
No full text審定書
授權書
中文摘要.............................................................. iii
Abstract .............................................................. iv
致謝...................................................................... v
目錄...................................................................... vi
第一章 研究背景與介紹 ........................................................................ - 1 -
第一節 草藥與微量元素介紹 ................................................................. - 1 -
第二節 臺南市售草藥及食用菇介紹 ...................................................... - 9 -
第三節 原子吸收光譜儀介紹 ............................................................... - 14 -
第四節 火焰式原子吸收光譜儀介紹 ..................................................... - 15 -
第五節 實驗目的 .................................................................................. - 18 -
第二章 材料與方法 ................................................................................ - 19 -
第一節 儀器設備介紹 ............................................................................. - 19 -
第二節 試劑 ............................................................................................ - 20 -
第三節 實驗器皿清洗 .............................................................................. - 20 -
第四節 實驗溶液配置 ............................................................................... - 21 -
第五節 草藥及菇類樣本處理 .................................................................... - 25 -
1.樣本清洗及乾燥 ..................................................................................... - 25 -
2.草藥沖泡液配置 ...................................................................................... - 25 -
3.草藥沖泡液的消化 .................................................................................. - 26 -
4.草藥和菇類的微波消化 ........................................................................... - 26 -
第六節 方法驗證 ....................................................................................... - 27 -
1.檢量線 ..................................................................................................... - 27 -
2.準確度 ..................................................................................................... - 27 -
3.精密度 ..................................................................................................... - 27 -
4.偵測極限 .................................................................................................. - 28 -
5.定量極限 .................................................................................................. - 28 -
6.汙染控制 ................................................................................................... - 28 -
第三章 結果與討論 ...................................................................................... - 29 -
第一節 草藥清洗方式比較 ............................................................................ - 29 -
第二節 最佳化條件 ....................................................................................... - 29 -
1.氣體流速和燃燒頭高度 ............................................................................... - 30 -
第三節 分析方法驗證 .................................................................................... - 30 -
1.檢量線 ......................................................................................................... - 30 -
2.準確度 ......................................................................................................... - 32 -
3.精密度 ......................................................................................................... - 35 -
4.偵測極限和定量極限 .................................................................................... - 36 -
第四節 分析方法應用 ....................................................................................... - 37 -
第四章 結論 ..................................................................................................... - 43 -
參考文獻 ........................................................................................................... - 44 -
圖表附錄 ........................................................................................................... - 49 -
表一、 草藥清洗方式比較 （Ⅰ：酒精、Ⅱ：蒸餾水；Mean±SD, ppm）........ - 49 -
表二、 火焰式原子吸收光譜儀元素最佳化條件 ................................................ - 50 -
表三、 魚腥草沖泡消化液回收率 ........................................................................ - 51 -
表四、 仙草沖泡消化液回收率 ............................................................................ - 53 -
表五、 魚腥草微波消化液回收率 ......................................................................... - 55 -
表六、 仙草微波消化液回收率 ............................................................................. - 57 -
表七、 金頂側耳菇微波消化液回收率 ................................................................. - 59 -
表八、 以火焰式原子吸收光譜儀測定鐵標準品精密度之結果 .......................... - 61 -
表九、 以火焰式原子吸收光譜儀測定銅標準品精密度之結果 .......................... - 62 -
表十、 以火焰式原子吸收光譜儀測定鈣標準品精密度之結果 .......................... - 63 -
表十一、 以火焰式原子吸收光譜儀測定鋅標準品精密度之結果 ....................... - 64 -
表十二、 以火焰式原子吸收光譜儀測定錳標準品精密度之結果 ....................... - 65 -
表十三、 以火焰式原子吸收光譜儀測定鎳標準品精密度之結果 ....................... - 66 -
表十四、 以火焰式原子吸收光譜儀測定鉛標準品精密度之結果 ....................... - 67 -
表十五、 以火焰式原子吸收光譜儀測定鎘標準品精密度之結果 .. .................... - 68 -
表十六、 火焰原子吸收光譜儀所測定各元素之LOD、LOQ ...............................- 69 -
表十七、 火焰式原子吸收光譜儀測定草藥沖泡液中元素的含量 ....................... - 70 -
表十八、 火焰式原子吸收光譜儀測定草藥微波消化液中元素的含量 ................ - 71 -
表十九、 火焰式原子吸收光譜儀測定菇類微波消化液中元素的含量 ................ - 72 -
表二十、 微量元素參考攝取量 ................................................................................ - 73 -
表二十一、 膳食營養素上限攝取量及食品衛生法中蔬果可食部分中金屬含量符合以下 限量 …………………………………...................................………………………........... - 74 -
表二十二、 食品衛生法中食用菇類（子實體）中金屬含量符合以下限量 ........ - 75 -
圖一、原子吸收光譜法儀器 ..................................................................................... - 76 -
圖二、中空陰極管（hollow cathode lamp, HCL） ............................................. - 77 -
圖三、層流式燃燒器 ................................................................................................. - 78 -[[abstract]]使用藥用植物做為療法或膳食補充劑可以追朔到以前，但在過去幾十年裡大幅增加使用。由於疾病和死亡的報告，最近被質疑其使用上的安全性。在亞洲、歐洲和美國也被報導出藥用植物存在毒性元素。藥用植物容易在種植時及加工過程中受到汙染。在植物中元素的含量受到地球上土壤和植物的選擇性累積一些元素影響。因其健康的目的，測定臺灣草藥、菇類和沖泡液，如：馬鞭草、魚腥草、仙草、金絲草、半邊蓮、黃花蜜菜、金頂側耳菇中七種礦物質及微量元素（鐵、銅、鋅、鎳、錳、鉛和鎘）。以火焰原子吸收光譜儀來測定草藥、菇類及沖泡液。草藥、菇類和沖泡液中其元素含量有很大的差異。鐵、銅、鋅、鎳、錳、鉛和鎘含量最高，分別是2779.5 mg/kg、32.5 mg/kg、1522.2 mg/kg、3.5 mg/kg、164.1 mg/kg、4.8 mg/kg、2.3 mg/kg。對於有好的藥材和菇類的品質管控是很重要的，以保護消費者遠離汙染。
The use of medicinal plants in therapeutics or as dietary supplements goes back beyond recorded history, but has increased substantially in the last decades. However, the safety of their use has recently been questioned due to the reports of illness and fatalities. Poisonings associated with the presence of toxic metals in medicinal plants were reported in Asia, Europe and the United States. Medicinal herbs may be easily contaminated during growing and processing. The level of essential elements in plants is conditional, the content being affected by the geochemical characteristics of the soil and by the ability of plants to selectively accumulate some of these elements. Seven mineral and trace elements (Fe, Cu, Zn, Ni, Mn, Pb, Cd) were determined in the herbs and mushrooms and their infusions consumed for health purposes in Taiwan. Element concentrations in the medicinal herbs and their infusions were determined by FAAS. The mineral and trace element content of medicinal herbs and their infusions was showed a wide variability. Fe, Cu, Zn, Ni, Mn, Pb, Cd was found in samples of the other medicinal herbs at levels up to 2779.5 mg/kg, 32.5 mg/kg, 1522.3 mg/kg, 3.5 mg/kg, 164.1 mg/kg, 4.8 mg/kg, 2.3 mg/kg, respectively. It is important to have a good quality control for herbal medicines and mushrooms in order to protect consumers from contamination
Study of the Role of Human PGC-1 alpha Protein Phosphorylation in Downstream Gene Expression and Neurite Outgrowth
No full textContents i
Figure contents iii
致謝辭 iv
Abbreviation v
中文摘要 1
Abstract 3
Chapter 1 Introduction
1-1 PGC family 5
1-2 PGC-1alpha 6
1-3 Nuclear respiratory factor 1 7
1-4 Roles of NRF-1 and PGC-1alpha in neurons 7
1-5 Phosphorylation of PGC-1alpha 9
1-6 Hypothesis and specific aims 9
1-6-1 Hypothesis 9
1-6-2 Specific aims 10
Chapter 2 Materials and Methods
2-1 Cell culture 11
2-2 Plasmid constructs 11
2-3 Transient transfection 12
2-4 Measurement of neurite outgrowth 13
2-5 Dual-luciferase assay 13
2-6 Statistical analysis 13
Chapter 3 Results
3-1 Mutation of PGC-1alpha affects neurite outgrowth 15
3-2 Mutation of PGC-1alpha alters the promoter activity of
NRF-1 downstream gene 16
3-3 Mutation of PGC-1alpha alters the promoter activity of
PPAR downstream gene 17
Chapter 4 Discussion
4-1 Phosphorylation sites of PGC-1alpha involve in neuronal
functions and transcriptional coactivation 18
4-2 Several phosphorylation sites involve in PGC-1alpha
activation 18
4-3 Protein kinases involve in PGC-1alpha activation 19
4-4 Conclusion 20
Chapter 5 References 21
Tables and Figures 27
Appendix 36
作者簡歷 41
Figure contents
Table 1. Possible phosphorylation sites of human PGC-1alpha 27
Table 2. Primers for constructing mutant PGC-1alphas on nucleotides
referring to phosphorylation sites 28
Table 3. Summary of functional activity of mutant PGC-1alphas 35
Figure 1. Possible phosphorylation sites of PGC-1alpha 29
Figure 2. Effects of mutant PGC-1alphas on neurite outgrowth 30
Figure 3. Effects of mutant PGC-1alphas on IAP promoter activity 32
Figure 4. Effects of mutant PGC-1alphas on UCP-1 promoter activity 33
Appendix 1. Human PGC-1alpha cDNA sequence 36[[abstract]]PGC-1共同活化因子家族當中包含PGC-1alpha、PGC-1beta以及PRC。先前的研究發現PGC-1alpha在肝臟、心臟以及骨骼肌肉細胞中扮演刺激粒線體呼吸作用及基因的轉錄功能，並且活化重要的轉錄因子，包含NRF-1與PPARgamma。然而，PGC-1alpha在轉譯後修飾的功能調控，例如磷酸化、乙醯化和甲基化的研究仍不清楚。但是在先前的研究發現，Akt和p38 MAPK 對PGC-1alpha蛋白磷酸化與該蛋白的活化有關。我們也預測出一些激酶在PGC-1alpha磷酸化的部位，包括PKC、PKA與MAPK。不過，目前還不清楚這些磷酸化位置是否會影響NRF-1下游基因的啟動子活性和神經突的生長。首先我們選殖完整的人類PGC-1alpha，接下來利用定點突變選殖突變的PGC-1alpha，編號為M1-14代表突變不同的可能磷酸化部位之PGC-1alpha。將這些質體轉殖到神經母細胞株IMR-32細胞以量測神經突，或轉殖到IMR-32及肝臟腫瘤細胞株HepG2細胞來量測啟動子活性。大量表現PGC-1alpha可以促進神經突生長，大量表現一些突變的PGC-1alpha會減少促進神經突生長的效果，如M1、4、8與10。分別表現PGC-1alpha在IMR-32與HepG2細胞，則會促進人類IAP及UCP-1基因啟動子活性，但表現一些突變的PGC-1alpha則會降低促進效果。如大量表現PGC-1alpha的M3、4及11-14，會抑制PGC-1alpha促進IAP基因啟動子活性，而M3、5-9及12-14則會抑制PGC-1alpha促進UCP-1基因啟動子活性。根據本研究所得的結果，PGC-1alpha蛋白磷酸化修飾對蛋白本身的功能扮演重要的角色，而且對功能的調控具有細胞與基因專一性。
PGC-1 coactivator family consists of PGC-1alpha, PGC-1beta and PRC. Previous studies have shown that the PGC-1alpha is a potent stimulator of mitochondrial respiration and gene transcription in liver, heart as well as skeletal muscle and coactivates transcriptional activity of nuclear proteins, including nuclear respiratory factor 1 (NRF-1) and peroxisome proliferator-activated receptor γ (PPARgamma). The functions of PGC-1alpha may be influenced by posttranslational regulation, such as phosphorylation, acetylation and methylation. Previous studies revealed that Akt and p38 MAPK phosphorylation sites of PGC-1alpha involve in its functional activity. We also predicted several phosphorylation sites for other kinases, such as PKC, PKA and MAPK. However, it is still unclear whether these sites and other putative phosphorylation sites affect the transcriptional activity of its downstream genes and neurite outgrowth. To address this issue, we cloned full-length wild type human PGC-1alpha and mutated some phosphorylation sites using site-directed mutagenesis. These mutant constructs were named M1 to M14. Subsequently, we transfected these constructs into cells to measure neurite length in human neuroblastoma IMR-32 cells and to determine promoter activity in IMR-32 cells and hepatoma HepG2 cells. Overexpression of wild type PGC-1alpha increased neurite outgrowth whereas that of mutant constructs, M1, 4, 8 and 10, decreased the induced neurite outgrowth. Overexpression of wild type PGC-1alpha also enhanced promoter activities of human IAP and UCP-1 genes. However, overexpression of M3, 4 and 11-14 mutants decreased the induced IAP promoter activity in IMR-32 cells. In addition, overexpression of M3, 5-9 and 12-14 mutants decreased the induced UCP-1 promoter activity in HepG2 cells. According to these results, phosphorylation of PGC-1alpha may play an important role in its functions and these effects are cell- and gene-specific
Isolation, Identification And Antioxidant Activity Of Feruloyl-L-Arabinose From Coba Husk
No full text致謝 ii
中文摘要 iii
Abstract vi
目錄 ix
研究動機 1
研究目的 2
第二章 文獻回顧 3
阿魏酸 3
阿魏酸寡醣 6
自由基 (Free Radical) 9
生物體內抗氧化系統 12
論文架構 16
第三章 材料與方法 17
實驗藥品與儀器 17
1. 脫脂茭白筍殼之製備 20
2. 茭白筍殼不可溶膳食性纖維製備 20
3. 茭白筍殼不可溶膳食性纖維酸水解之製備 21
3.1 實驗設計 21
3.1.1 最適作用溫度探討 21
3.1.2最適作用 TFA酸濃度探討 22
3.1.3 最適作用時間探討 22
4總溶解阿魏酸及游離阿魏酸測定 22
4.1 游離阿魏酸含量測定 23
4.2 總溶解阿魏酸含量測定 23
4.3 酯化阿魏酸含量測定 24
5. 反應曲面法的設計 ( Response Surface Methodology; RSM ) 24
6. 阿魏酸寡醣( Feruloyl oligosaccharides ; FAO )的劃分及純化 25
6.1 高效率液相層析儀純化 ( High-performance Liquid Chromatograph; HPLC ) 26
7. Feruloyl-L-arabinose(FAA)醣類的鑑定。 28
8. 抗氧化分析 29
8.1 ABTS自由基清除能力測定 29
8.2 以DNA電泳分析OH自由基造成DNA傷害試驗 31
8.3氧自由基吸收能力分析法(Oxygen-radical absorbance capacity; ORAC) 32
8.4 超氧陰離子自由基清除能力測定(Superoxide radical scavenging assay) 33
第四章 結果與討論 35
1. 不同單因子對酯化阿魏酸生成的影響 35
1.1 作用溫度對酯化阿魏酸生成影響 35
1.2 作用酸濃度對酯化阿魏酸生成影響 36
1.3作用時間對酯化阿魏酸生成影響 36
2. 反應曲面法(response surface methodology,RSM) 37
3. 茭白筍殼不可溶膳食性纖維中酯化阿魏酸的分離及純化 38
4. 結構鑑定 39
5. 抗氧化能力分析 41
5.1 ABTS自由基清除能力探討 41
5.2 以DNA電泳評估OH自由基清除能力探討 41
5.3 ORAC氧自由基吸收能力分析 42
5.4 超氧陰離子自由基清除能力 43
第五章 結論 45
附錄 66
參考文獻 68
圖目錄
圖1. 作用溫度對酯化阿魏酸生成的影響 47
圖2.作用TFA酸濃度對酯化阿魏酸生成的影響 48
圖3.作用時間對酯化阿魏酸生成的影響 49
圖4. 不同酸濃度與不同時間酸水解交互作用對酯化阿魏酸生成 的反應曲面及等高線圖。 53
圖5. 阿魏酸寡醣以甲醇萃取後HPLC圖譜分析 54
圖6. Feruloyl-L-arabinose(FAA) 13C碳譜圖 55
圖7. Feruloyl-L-arabinose(FAA) 1H碳譜圖 56
圖8. Feruloyl-L-arabinose(FAA) 1H-13C COSY光譜圖。 57
圖9 . Feruloyl-L-arabinose(FAA) EI-MS質譜圖 58
圖10 . Feruloyl-L-arabinose(FAA)的醣類鑑定 59
圖11 .Feruloyl-L-arabinose(FAA)結構圖 60
圖12 . Ferulic acid(FA)、Feruloyl-L-arabinose(FAA)、Trolox ABTS自由基清除能力分析。 61
圖13 .Ferulic acid(FA)、Feruloyl-L-arabinose(FAA)對DNA保護能 力分析 62
圖14 .Ferulic acid(FA)、Feruloyl-L-arabinose(FAA)、Trolox ORAC氧自由基吸收能力分析。 64
圖15 .Ferulic acid(FA)、Feruloyl-L-arabinose(FAA)、Vit C 超氧陰離子自由基清除能力分析。 65
表目錄
表1. 中心混成設計(central composite design, CCD)中因子的編碼 及實際值對照表 50
表2. 依中心混成實驗設計所得到的酯化阿魏酸含量 51
表3. 中心混成設計實驗所得酯化阿魏酸含量之廻歸分析 52
表4. Protection against oxidative damage to PGEX-4T-2 by FAA and FA 63[[abstract]]將農業廢棄物轉變成可利用的物質，不僅可減少環境污染更可提升經濟價值。阿魏酸 (ferulic acid, FA)是植物細胞壁中含量豐富的酚酸，其生理功能已被廣泛研究。近年來阿魏酸寡醣被發現不僅具有優秀的抗氧化功能，亦有與寡醣類一樣促進腸道有益菌增生的效果。農業廢棄物皆含植物細胞壁，基於廢棄物回收利用，本研究先擬由台灣埔里盛產的茭白筍殼廢棄物進行酸水解來探討阿魏酸寡醣的製備。
首先以單因子依序探討作用溫度、酸(TFA)濃度及作用時間對酯化阿魏酸生產的影響。實驗結果分別顯示於100℃，0.2 M TFA及作用1小時會有最高酯化阿魏酸的產生。由於水浴溫度的限制，將溫度固定於100℃，設定酸濃度及作用時間為因子，進一步利用反應曲面法之中心混成試驗來進行最適生產條件的探討，由10組實驗設計所得到的結果經分析後(R2=0.95)，酸濃度及作用時間皆是顯著影響因子且具交互作用，經由此模式所預測之最適生產條件為於100℃下，加入 0.234 M TFA作用59.9 min可得5.583mg/g 酯化阿魏酸。於確認實驗中，於100℃下，加入 0.234 M TFA作用60 min可得5.626mg/g 酯化阿魏酸，由此可證明此模式的適用性。
並利用上述最適條件所得之水解液進一步進行阿魏酸寡醣的製備。水解液經XAD-2陰離子管柱劃分後，約64%酯化阿魏酸被發現於50%甲醇沖提液中，將此部分經凍乾後，利用甲醇與水依序進行partition，可發現有84%之酯化阿魏酸於甲醇萃取液，經HPLC初步分析，發現有一單獨且主要的波峰(占總迴歸面積之37%)於24.43分鐘出現，因此進一步利用半製備型HPLC管柱進行分離純化，收集所得物質並經核磁共振(NMR)及質譜儀(EI)光譜分析後比對相關文獻，所得物質證實為Feruloyl-L-arabinose (FAA)。
進一步探討其對不同活性氧自由基(reactive oxygen species, ROS)的抗氧化能力。於總抗氧化能力探討中，其清除ABTS 自由基能力IC50為15.86 μg/ml；於經由Fenton reaction 所產生之OH自由基對DNA斷裂的保護效果上，5μg /ml FAA與25μg /ml FA所展現的保護效果一致；而在ORAC (oxygen radical absorbance capacity) 氧自由基吸收能力中，0.25μg/ml FAA 清除AAPH自由基的能力與市售抗氧化劑Trolox(6.25μg/ml)相當，而0.5μg/ml FAA清除AAPH自由基的能力與0.25μg/ml FA相當；在超氧陰離子自由基清除能力中，FAA 所展現之清除效能並不顯著，其IC50為95.04 μg/ml，低於FA (39.02 μg/ml)與ascorbic acid (13.67 μg/ml)。基於上述結果可發現FAA與FA對過氧自由基 (peroxyl radical)與OH自由基之清除能力較佳，且FAA的清除能力約為FA的一半，由其分子量的比較顯示FAA的抗氧化功能應來自FA部分；但FAA不僅與FA一樣可溶於甲醇，更可溶於水，因此FAA基於其抗氧化能力所可應用的領域較FA更廣，特別是在食品工業的應用。
Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is an abundant phenolic acid that is present in the plant cell wall and has an important role in linkage of hemicellulosic polysaccharides with other cell wall components. Ferulic acid with several potential industrial and medical applications had reported elsewhere. Recently, water-soluble ferulic acid sugar esters from wheat bran had been reported that can stimulate the growth of Bifidobacterium bifidum, and also show excellent antioxidant activity in vitro and in vivo studies. Based on the regard of agricultural wastes re-newly use, conversion of agro-industrial residues to ferulic acid sugar esters not only provides an alternative substrate but also helps to solve some of the problems caused by their accumulation. Coba husk, that abundantly generated from Pu Li, Taiwan, was selected as substrates for ferulic acid sugar esters preparation by acid hydrolysis in this work.
The classical “change-one-factor-at-a-time” approach was first employed to evaluate the effects of hydrolysis conditions, including incubation temperature, TFA concentration and incubation time, on esterified ferulic acid (EFA) producing. Results show the maximum condition for EFA producing was at 100℃, 0.2 M TFA and duration time of 60 min, respectively. A 23 central composition design (CCD) of response surface methodology (RSM), which factors of TFA concentration and incubation time were selected, was further introduced to optimize the condition for EFA preparation. Data from the 10 sets were analyzed to yield regression equations and regression coefficients (R2). From the response surface regression (RSREG) data, the determination coefficient R2 = 0.95 and all variables significantly contributed to this effect. The maximum EFA production predicted by this model is 5.583mg/g from canonical analysis of response surface under the conditions of 0.234 M TFA, at 100℃ and incubated for 59.9 min. After verified the optimum condition, 5.626mg/g EFA were obtained and demonstrated the applicability of this model.
Esterified ferulic acids were prepared based on the above optimal condition. After fractionated by an Amberlite XAD-2 column, fraction obtained by eluting with 50% methanol/water was found to possess 64% EFA recovery. After lyophilized, sample was extracted sequential with methanol and H2O. Approximately 84% EFA of 50% methanol/water elute was found in MeOH extract, and then used for further separation. A preliminary survey of the chromatographic profile of this fraction by reverse phase HPLC analysis using a mobile phase of 20% MeOH/H2O, a major peak with retention time at 24.43 min was observed that comprise 37% of total regression area. In order to isolate this compound, re-chromatographed was conducted by semi-preparative reversed-phase HPLC. A pure compound was isolated as a light yellow powder and identified as feruloyl-L-arabinose (FAA) after intensive NMR and EI mass spectrum analysis and compared to earlier reports.
The antioxidant activity, including total antioxidant activity and toward different reactive oxygen species, of FAA was further investigated. In the TEAC assay, FAA show the 50% inhibition concentration (IC50) value of FAA was 15.86 μg/mL in comparison with the standard antioxidants FA and Trolox with IC50 values at 2.96 and 4.76μg/mL, respectively. The super coil (SC) form in DNA can be converted to the open circular (OC) form due to the hydroxyl radical damage which generated based on the Fenton reaction. In evaluating the protection effect of FAA on oxidation-induced DNA damage study, FAA and FA both exhibited the protective capacity 34.6%~56.6% and/or 58.3~64.4% in the concentration range of 2.5~10μg/mL, respectively. The ORAC assay was further used to test the antioxidative capacity of antioxidants to quench peroxyl radicals. In ORAC assay, FAA had a concentration-dependent increase in the inhibition of fluorescein decay. The antioxidative activity against peroxyl radicals was in the order of FA> FAA > Trolox in this study. This demonstrated FAA could directly scavenge the peroxyl radicals to terminate the free radical chain reaction of lipid peroxidation. Superoxide radicals could promote oxidative reactions and form perhydroxyl radicals which initiate lipid oxidation. FAA could effectively scavenge superoxide radicals (IC50 95.04 ug/mL), however, less than ascorbic acid (IC50 13.67 ug/mL) and FA (IC50 39.02 ug/mL). Based on the above results, FAA and FA can exhibit significantly antioxidant activity especially in peroxyl radical and OH radical scavenging. The antioxidant capacity of FAA slightly less than FA should attribute to its sugar moiety. However, the sugar moiety makes FAA more hydrophilic than FA. Based on this character, FAA should superior than FA that can adapt to industrial application, especially in food industry
The Study of Chitosan Used for Bacteriostatin in Cosmetics
No full text目錄
中文摘要......................................................................2
英文摘要......................................................................4
表目錄........................................................................8
圖目錄........................................................................9
縮寫表........................................................................10
第一章 前言....................................................................11
第二章 研究背景................................................................12
2.1研究目的與動機..............................................................12
2.2研究架構...................................................................12
2.3幾丁質與幾丁聚醣簡介.........................................................13
2.3.1高分子材料應用領域.........................................................17
2.3.2幾丁聚醣應用領域...........................................................17
2.3.3幾丁聚醣的抑菌機制.........................................................18
2.3.4化妝品中常見抑菌劑.........................................................18
第三章 材料與方法............................................................................19
3.1實驗藥品....................................................................19
3.1.1實驗菌種與細胞 ............................................................20
3.1.2幾丁聚醣之製備.............................................................20
3.1.3乳酸之製備.................................................................23
3.1.4抑菌實驗對照組之製備........................................................23
3.1.5實驗藥品製備...............................................................24
3.2實驗儀器設備.................................................................24
3.3實驗方法....................................................................25
3.3.1最低抑菌濃度測定 ( MIC) ..................................................25
3.3.2細胞存活率(MTT assay)試驗.................................................25 3.3.3 細胞毒性(LDH assay)......................................................27 3.3.4重金屬檢驗................................................................29
3.3.5 微生物檢驗...............................................................31
第四章 結果與討論...............................................................32
第五章 結論....................................................................63
第六章 參考文獻................................................................65[[abstract]]本研究之目的主要是探討幾丁聚醣在化妝品中之抑菌能力，分別以不同濃度、不同分子量及pH值測定其抑菌效果，篩選出最佳抑菌濃度及pH值，並進行細胞存活率試驗、細胞毒性試驗、重金屬檢驗及微生物檢驗等試驗。
實驗選用樣品為幾丁聚醣，主要是因其溶解度較幾丁質高。幾丁聚醣不具毒性且具有良好的生物相容性與生物可分解性，對環境友善的高分子材料，在本篇研究中，我們選用三種不同分子量的幾丁聚醣，將幾丁聚醣(分子量100,000~200,000 datons)分別配製成0.5% ( pH 3.2和pH 5.6 )、1% ( pH 3.3和pH 5.7 )及1.5% ( pH 5.7 )，幾丁聚醣(分子量100,000~300,000 datons)濃度配製成0.5% ( pH 5.7 )、1% ( pH 5.7 )及2% ( pH 3.4 )，幾丁聚醣(分子量200,000~400,000 datons)濃度配製成1% ( pH 5.7 )，並以乳酸為溶劑，使幾丁聚醣分子量降解，藉此改善幾丁聚醣的溶解性質後，進行抑菌試驗分析，實驗結果發現幾丁聚醣(分子量100,000~200,000 datons)在濃度為0.5%且pH值為5.6時，對大腸桿菌、金黃色葡萄球菌均具有抑菌效果，而幾丁聚醣(分子量100,000~300,000 datons)與幾丁聚醣(分子量200,000~400,000 datons)的抑菌效果較差；同時，將濃度為0.5% (pH 5.6)、1% (pH 5.7)的幾丁聚醣(島久試藥)和濃度為1% (pH 5.7)的幾丁聚醣(分子量100,000~300,000 datons)與乳酸(對照組)及化妝品中常見抑菌劑(Chlorhexidine Gluconate、Microcare MTI)進行細胞存活率(MTT)及細胞毒性(LDH)試驗，實驗數據結果發現，乳酸(對照組)及化妝品中常見抑菌劑皆具細胞毒性且細胞無存活，然而不同濃度的幾丁聚醣對細胞具有良好存活率且不具細胞毒性。
將濃度為1.5%，pH 5.7的幾丁聚醣(分子量100,000~200,000 datons)進行重金屬檢驗，實驗結果為ND，此即表示幾丁聚醣不含重金屬元素。另外，將配製濃度為1 %、pH 5.6和0.5%、pH 5.6及1.5 %、pH 5.7的幾丁聚醣進行微生物檢測，結果顯示本研究中之幾丁聚醣之綠膿桿菌、大腸桿菌、金黃色葡萄球菌及總生菌數的檢測值為ND ，此即表示本研究之樣品不含上述之菌種(表示未檢出)。
Purpose of this study was to investigate chitosan in cosmetics in the antimicrobial activity, respectively, at different concentrations and different molecular weight and pH were measured inhibitory effect, filter out the best inhibitory concentration and pH, and the cell survival rate test, cytotoxicity test, heavy metal testing and microbiological testing and other tests.
Experimental use of sample to chitosan is mainly because of its better solubility than chitin. Chitosan non-toxic and biocompatible with Biodegradable, environmentally friendly polymer materials. In this study, we used three different molecular weight chitosan, the chitosan (MW:100,000~200,000 datons) were prepared as 0.5% (pH 3.2 and pH 5.6)、1% (pH3.3 and pH5.7) and 1.5% (pH5.7) chitosan (MW:100,000~300,000 datons) concentration of the preparation of 0.5% (pH 5.7), 1% (pH 5.7) and 2% (pH3.4) chitosan (MW:200,000~400,000 datons) concentration of the preparation into 1% (pH 5.7), and lactic acid as the solvent, the degradation of chitosan molecular weight, to improve the dissolution properties of chitosan the antibacterial agents antibacterial test analysis, experimental data and found that chitosan (MW:100,000~200,000 datons) at a concentration of 0.5% and the pH value of 5.6, that for Escherichia coli, Staphylococcus aureus all have antibacterial effects, the chitosan and chitosan (MW:200,000~400,000 datons) the inhibitory effect of poor; the same time, the concentration of 0.5%(pH5.6), 1%(pH5.7) chitosan (MW:100,000~200,000 datons) and a concentration of 1% (pH 5.7) of the chitin polymer sugar and lactic acid (control group) and a common antibacterial agent of cosmetics for cell survival and cell toxicity test, experimental results showed that lactic acid (control group) and common cosmetics and antibacterial agent with a cytotoxic cell-free survival, and different concentrations of chitosan on cell survival (MTT) and do not have good cytotoxicity (LDH).
The concentration of 1.5%, pH 5.7 chitosan (MW:100,000~200,000 datons) for heavy metals testing, test data results for the ND, namely, that chitosan does not contain heavy metals. In addition, the preparation of a concentration of 1%, pH 5.6 and 0.5%, pH 5.6 and 1.5%, pH 5.7 chitosan for microbiological testing, results showed that chitosan on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and total plate count detection is ND, namely, that the study sample dose not contain the above strains (that is not detected)
Evaluation of Chinese Herbal Water Extracts in Regulating Adipose Accumulation
Get PDF中文摘要………………………………………………………………………ii
英文摘要 ……………………………………………………………………iii
壹、導論…………………………………………………………………………1
貳、研究構想………………………………………………………………16
參、材料與方法…………………………………………………………18
肆、結果………………………………………………………………………29
伍、討論與結論……………………………………………………………35
陸、參考文獻………………………………………………………………39
柒、表……………………………………………………………………………39
捌、圖……………………………………………………………………………51
玖、附表…………………………………………………………………………50
拾、附圖…………………………………………………………………………51[[abstract]]近年來，肥胖已成為全球重視的一項公共衛生問題。肥胖是罹患心血管疾病、糖尿病與高血壓等疾病的危險因子。從能量平衡的角度來看，肥胖被認為是多餘的能量以脂肪的形式累積於脂肪組織的情形，因此造成體重逐漸增加。許多研究指出天然植物中所含的的化學成分具有抑制脂解酵素、α-澱粉酶與α-葡萄糖苷酶的功能。而利用中草藥的天然成分來改善人體健康已受到相當的重視。本研究從坊間中草藥店隨機選取57種中草藥作為實驗材料，以水萃取之方式，經由冷凍乾燥濃縮後，利用抑制酵素分析方法從中選取具有抑制脂解酵素、α-澱粉酶與α-葡萄糖苷酶能力的中草藥。經抑制酵素實驗結果發現，57種中草藥大部分對三種酵素都有抑制的效果，有少部分對於酵素有促進活性的效果，而E14中草藥對此三種酵素有最佳抑制效果。E14以濃度0.15625 X進行抑制實驗有最佳效果，對脂解酵素、α-澱粉酶與-葡萄糖苷酶的抑制效果分別為80、106與94%。為瞭解萃取物與酵素間之作用關係，使用E14萃取物進行酵素競爭試驗，發現E14與脂解酵素及α-澱粉酶之間的關係是屬於非競爭型抑制，與-葡萄糖苷酶則為不競爭型。總多酚含量實驗發現，E14在57種中草藥之中，並不是總多酚含量最多之中草藥。為探討何種成分具抑制作用，進而以HPLC去分析E14中草藥萃取物中的成分。HPLC分析實驗選用C18管柱去分析E14中草藥萃取物，並以波長210 nm 為偵測波長，每分鐘收集分層液，再以不同分層液進行酵素抑制實驗。結果發現，在53-54 min 這個分層液對脂解酵素、α-澱粉酶與α-葡萄糖苷酶之抑制率分別為59.36 98.81與95.02%。由以上結果可得知E14這個中草藥之水萃取物，可以抑制脂解酵素、α-澱粉酶與α-葡萄糖苷酶，其抑制能力與E14中草藥中所含的特定成份有關係。
In recent years, obesity has become a sanitary problem that the global people put emphasis on. Obesity is a bad factor associated with cardiovascular diseases, diabetes, high blood pressure, and so on. Because of imbalance of intake and expenditure, obesity is thought of the condition that the excess energy is converted into fat, which is then accumulated in adipose tissues. Therefore, it causes gradual weight gain. Many studies showed that chemicals in the natural products may function in inhibiting lipase, α-amylase and α-glucosidase to decrease lipid accumulation. Recently, it is greatly emphasized on using the natural chemicals in the Chinese herbs to help improve people health. In this study, we collected fifty seven Chinese herbs from stores as materials. We used freeze-dried concentrated water extracts of herbs and the enzyme inhibition analysis to search for Chinese herbs that inhibit lipase, α-amylase and α-glucosidase. The results of enzyme inhibition analysis showed that most Chinese herbs represent inhibiting effects on these three enzymes, whereas a few herbs have adverse effects, increasing the activity of some enzymes. One of the fifty seven Chinese herbs, E14, exerts most inhibition activity on these enzymes. When using dilution factor of 0.15625X, the inhibition rates of lipase, α-amylase and α-glucosidase are 80, 106 and 94%, respectively. To address whether the inhibition is competitive, noncompetitive or uncompetitive, we performed competitio tests and found that the inhibition effects of E14 on lipase and α-amylase are uncompetitive and, however, effect on α-glucosidase is noncompetitive. The measurements of total polyphenol content of these herbs showed that E14 is not the richest. Therefore, we used the HPLC to analyze the ingredients of Chinese herbal extracts. We used C18 column and ultraviolet–visible spectroscopy on wavelength of 210 nm to detect the ingredients. Then we collected the analyzed fraction per minute for the enzyme inhibition experiments. The results showed that fraction during 53-54 min has 59.36, 98.81 and 95.02% of inhibition rates on lipase, α-amylase and α-glucosidase, respectively. The conclusion is that the water extract of Chinese herb E14 can inhibit lipase, α-amylase and α-glucosidase and the functions are associated with some of its ingredients
Study on Growth Inhibition and Induction of Apoptosis by Methanol Extract of Antrodia Camphorata in MCF-7 Breast Cancer Cells
No full text授權書.................................................................................................I
目錄....................................................................................................III
圖目錄................................................................................................V
致謝....................................................................................................VII
中文摘要.............................................................................................X
Abstract...............................................................................................XII
I.前言
1.1 背景資料......................................................................................1
1.1.1 乳癌的病理機制.......................................................................1
1.1.2 台灣牛樟芝...............................................................................4
1.1.3 細胞凋亡...................................................................................5
1.2 研究目的......................................................................................7
II.方法與材料
2.1 材料..............................................................................................8
2.2 台灣牛樟芝的萃取與製備..........................................................9
2.3 細胞培養和與細胞存活率分析.................................................10
2.3.1 人類乳癌細胞株......................................................................10
2.3.2 細胞冷凍與解凍......................................................................10
2.3.3 繼代培養..................................................................................11
2.3.4 細胞總蛋白萃取......................................................................11
2.3.5 細胞毒性試驗..........................................................................11
2.4 流式細胞技術試驗.....................................................................13
2.5 西方墨點分析法.........................................................................15
2.6 統計學分析................................................................................16
III.結果
3.1 牛樟芝萃取物對於MCF-7細胞的生長與存活力影響.............17
3.2 流式細胞技術測定MCF-7細胞的存活力.................................19
3.3 牛樟芝萃取物對於細胞色素c釋放，caspase 3 活化，和 PARP 裂解的響..............................................................................................20
3.4 牛樟芝萃取物對於Bcl-XL protein的影響...............................21
IV. 討論與結論......................................................................22
參考文獻.........................................................................................25
圖與表.............................................................................................30
附錄.................................................................................................38[[abstract]]在台灣，牛樟芝是一種非常有名的中國傳統食用菌類藥物，近年來有研究指出，它具有抗氧化和抗癌功用。然而近幾年來台灣牛樟芝最近以來已經應用在中國傳統醫學上，以治療食物及藥物中毒，腹瀉，高血壓和肝癌等，但是目前卻很少有生物活性的試驗報告。再者，細胞凋亡和癌症之間的關係受到相當多的重視，有越來越多相關研究顯示腫瘤轉化、發展和轉移的相關過程涉及改變正常的細胞凋亡途徑。
台灣的婦女中，乳癌是最常見的惡性腫瘤。在乳癌中大約有三分之一的患者發展轉移，最後進展為乳癌末期。儘管如此現在對於早期乳癌的治療還是使用化療的方式。乳癌就像其他癌症一樣，由於環境和有缺陷的基因之間的相互作用產生後，正常的細胞會受限於細胞週期，當細胞發生不正常時細胞會啟動修復及凋亡機制，但是癌症細胞不受到細胞週期的限制，不會啟動修復及凋亡機制，進而產生癌化細胞。在美國，有10％至20％乳癌和卵巢癌患者，其一級或二級親屬有這些疾病，這些癌症的家族遺傳傾向，被稱為遺傳性乳腺癌 - 卵巢癌綜合徵。
將培養的MCF-7乳癌細胞加入不同濃度的台灣牛樟芝萃取物（5-20 μg/ml），因此MCF-7細胞開始產生細胞凋亡，染色質濃縮， 甚至DNA序列片段. sub G1階段累積。此外，在MCF-7乳癌細胞凋亡的過程中，將伴隨著細胞色素c釋放，caspase 3 活化，和 PARP的裂解。研究顯示，牛樟芝萃取物透過誘導細胞凋亡，抑制MCF-7乳癌的細胞生長，它未來可能將具有抗癌藥物產品中的高度應用價值。
In Taiwan, Antrodia camphorata is a very famous Chinese traditional medicine, in recent years, studies have pointed out that it has antioxidant and anti-cancer function. Taiwan Antrodia camphorata, however, recently has been applied in traditional Chinese medicine and used to treat food poisoning, diarrhea, high blood pressure and liver cancer, but very few biological activity test report. In recent years, the relationship between apoptosis and cancer are quite a lot of attention, more and more studies have shown that tumor transformation, progression and metastasis process involves changing the normal apoptotic pathway.
Breast cancer is the most common malignancy in Taiwan women. Approximately one-third of all women with breast cancer develops metastases and ultimately expires due to the effects of the disease. Despite the fact that many tumors initially respond to chemotherapy, breast cancer cells can subsequently survive and gain resistance to the treatment. Breast cancer, like other cancers, occurs because of an interaction between the environment and a defective gene. Normal cells divide as many times as needed and stop. They attach to other cells and stay in place in tissues. Cells become cancerous when mutations destroy their ability to stop dividing, to attach to other cells and to stay where they belong. In the United States, 10-20% of breast and ovarian cancer patients, one or two relatives with these diseases, these cancers familial tendency, known as hereditary breast cancer - ovarian cancer syndrome.
Antrodia camphorata (5-20 μg / ml) the cultured MCF-7 breast cancer cells with different concentrations of the MCF-7 cells undergo apoptosis, chromatin condensation, and even DNA sequence fragment accumulation of sub G1 stage.Further, in the process of apoptosis in MCF-7 breast cancer cells, will be accompanied by cytochrome c activation Caspase 3 of release, and PARP degradation.Studies have shown that Antrodia camphorata through induction of apoptosis, inhibit the growth of MCF-7 breast cancer cells, it may be the high value products with anti-cancer drugs
以電解混凝技術去除水溶液中聚乙烯醇及其COD之研究
No full text[[abstract]]聚乙烯醇(PVA)是一種用途相當廣泛之水溶性高分子聚合物，其廣泛應用於紡織漿料、黏合劑、薄膜等方面。每年排放含PVA之廢水約有650,000噸，若無法有效處理，不僅會對人體健康造成危害，更可能影響生態環境
Role of the Src Signaling Pathway in High Glucose-Induced Renal Proximal Tubular Hypertrophy
No full text中文摘要-----------------------------------------------------------------------------------i
英文摘要---------------------------------------------------------------------------------iii
圖表目錄---------------------------------------------------------------------------------vii
第一章：緒論--------------------------------------------------------------------------------1
1. 糖尿病----------------------------------------------------------------------------------2
2. 糖尿病腎病變-----------------------------------------------------------------------------3
(1). 葡萄糖與糖尿病腎病變的關係-----------------------------------------------------------5
(2). 糖尿病患的氧化傷害------------------------------------------------------------------7
(3). 糖尿病患體內之抗氧化防禦系統----------------------------------------------------------8
(4). 高糖所誘發的氧化壓力機制------------------------------------------------------------------8
3. 氧化壓力對DN的影響------------------------------------------------------------------------10
(1). Probucol和抗氧化劑NAC之間的關係-----------------------------------------------------11
4. PI3K訊息傳遞與糖尿病的調節作用--------------------------------------------------------------11
5. Src對糖尿病調節作用-----------------------------------------------------------------------12
(1). Src在氧化壓力所扮演的角色-----------------------------------------------------------14
(2). Na+-K+ ATPase/Src 複合體--------------------------------------------------------------16
(3). Src與細胞凋亡的關係---------------------------------------------------------------------16
6. Ets-1參與的調控機制-----------------------------------------------------------------------17
7. 細胞週期調控因子--------------------------------------------------------------------------18
(1). Cyclin and Cyclin-dependent kinases (cdk)--------------------------------------------18
8. 細胞週期與糖尿病腎病變的相關性---------------------------------------------------------------20
9. Dmp1與Arf-p53相關調控---------------------------------------------------------------21
10. 細胞凋亡---------------------------------------------------------------------------23
(1). Bcl-2---------------------------------------------------------------------------------23
(2). Caspases------------------------------------------------------------------------------24
(3). 細胞凋亡的活化路徑------------------------------------------------------------------------24
11. 探討腎近曲小管的重要性的研究顯示腎病變---------------------------------------------------------25
12. 研究目的---------------------------------------------------------------------------------25
第二章：材料與方法-----------------------------------------------------------------------------28
1. 藥品試劑----------------------------------------------------------------------------------29
2. 細胞培養與細胞冷凍保存-------------------------------------------------------------------31
3. 細胞計數-------------------------------------------------------------------------------32
4. 葡萄糖與甘露糖的製備---------------------------------------------------------------------32
5. 細胞溶解物的製備------------------------------------------------------------------------32
6. 細胞蛋白質總量的測定---------------------------------------------------------------------33
7. 硫酸十二酯鈉-聚丙烯醯胺凝膠電泳 -----------------------------------------------------------34
8. 西方墨點法分析 -------------------------------------------------------------------------34
9. 細胞毒性分析----------------------------------------------------------------------------35
10. 細胞生長速率 (MTT Assay Kit) -----------------------------------------------------------36
11. 抗氧化分析的測定(GST Assay) -------------------------------------------------------------37
12. 細胞過度肥大的分析------------------------------------------------------------------------38
13. 統計分析--------------------------------------------------------------------------------39
第三章：結果------------------------------------------------------------------------------------40
第四章：討論------------------------------------------------------------------------------------47
第五章：圖表------------------------------------------------------------------------------------52
第六章：參考文獻---------------------------------------------------------------------------------72
圖表目錄
SCHEME 1. ROS generation mechanisms in type 2 diabetes.----------------------------------------6
SCHEME 2. Schematic representation of the interplay between AGE and RAGE and high glucose in promoting mitochondrial superoxide production in the diabetic kidney.------------------------------------9
SCHEME 3. Probucol structure.-----------------------------------------------------------------11
SCHEME 4. The Src signaling pathway.----------------------------------------------------------14
SCHEME 5. Proposed mechanism for the role of sustained ROS-mediated EGFR activation in mediation of progressive tubulointerstitial injury.--------------------------------------------------------------------16
SCHEME 6. Schematic representation of a model postulating the specific regulation of S/M by CDK4.-------------------------------------------------------------------------------------------------------------21
SCHEME7. The DMP1 signaling pathway.----------------------------------------------------------22
SCHEME 8. The INK4-ARF signaling network.-----------------------------------------------------23
SCHEME 9. To investigate the effects of HG-induced renal tubular cell hypertrophy on ROS and the Src signaling pathway and antioxidant prubucol in human renal tubular epithelial cells.---------------------27
SCHEME 10. Effects of HG on Signaling Pathways, Cell Cycle, Hypertrophy Index and apoptosis Pathways in HK-2 cell.-----------------------------------------------------------------------------------------52
Fig. 1 Effects of HG and HM on cellular growth in human proximal tubular (HK-2) cells.--------53
Fig. 2 Effects of HG and HM on cellular mitogenesis in HK-2 cells.----------------------------54
Fig. 3 Effects of HG and HM on cellular morphology in HK-2 cells.-----------------------------55
Fig. 4 Effects of HG and HM on cytotoxicity and caspase 3 activity in HK-2 cells.-------------56
Fig. 5 Effects of HG on apoptosis regulatory molecules in HK-2 cells.-------------------------57
Fig. 6 Time course of Src, PI3K, ERK, JNK, and p38 MAPK protein activation in HK-2 cells.-----58
Fig. 7 Effect of HG on PI3K activation in HK-2 cells.-----------------------------------------59
Fig. 8 Effects of Src inhibitor, ERK inhibitor and the antioxidant probucol on HG-mediated ERK activation in HK-2 cells.-----------------------------------------------------------------------------------60
Fig. 9 Effects of some kinase inhibitors on HG-mediated JNK and p38 MAPK activation in HK-2 cells. -----------------------------------------------------------------------------------------------------------61
Fig. 10 Effects of HG, some kinase inhibitors, and HM on cell growth in HK-2 cells.-----------62
Fig. 11 Effects of HG, some kinase inhibitors, and HM on cytotoxicity in HK-2 cells.-----------63
Fig. 12 Effects of HG and HM on GST activity in HK-2 cells.------------------------------------64
Fig. 13 Effects of Src inhibitor and the antioxidants on HG-inhibited GST activity in HK-2 cells.--------------------------------------------------------------------------------------------------------------65
Fig. 14 Effects of ERK inhibitor, Raf-1 inhibitor, and Src inhibitor on HG-mediated Ets-1, p-B-myb, cdk4, cyclin D1, Dmp1 and p53 expression in HK-2 cells.-----------------------------------------------------66
Fig. 15 Effects of some kinase inhibitors and the antioxidant probucol on HG-modulated cellular morphology in HK-2 cells.------------------------------------------------------------------------------------67
Fig. 16 Effects of Src inhibitor, ERK inhibitor and the antioxidant probucol on HG-induced cellular hypertrophy in HK-2 cells.----------------------------------------------------------------------------------68
Fig. 17 Effects of some kinase inhibitors and the probucol on HG-mediated collagen IV, and p27 protein expression in HK-2 cells.-----------------------------------------------------------------------69
Fig. 18 Effect of HG on cellular growth and hypertrophy in human renal proximal tubular cells.--70[[abstract]]糖尿病腎病變是造成我國透析患者需要洗腎的主要原因之一。糖尿病腎病變的研究主要集中在探討腎細胞過度肥大、增生和細胞外基質擴張，以及進一步引發的腎絲球或腎小管組織間隙纖維化和末期腎病。而上述的發展過程有牽扯到兩個關鍵調節者包括高葡萄糖和活性氧的衍生物。高葡萄糖會增加氧化壓力，促進細胞外基質蛋白質的合成和腎小管組織間隙纖維化，並產生一些對腎細胞的毒性作用。而腎小管肥大可能是一開始就會出現的病變過程，最終它將導致腎小管間質纖維化及糖尿病腎病變。儘管如此，在糖尿病腎病變中抗氧化劑對高葡萄糖誘發氧化壓力與腎小管肥大之作用機轉，目前仍然不清楚。
本研究中我們將測定高葡萄糖對腎小管細胞生長的影響以及Src/ERK訊息傳導途徑在調控細胞過度肥大方面所扮演的角色。結果發現葡萄糖呈現劑量依賴性抑制人類腎小管上皮細胞之生長。其次，高葡萄糖(500 mg/dl)可顯著增強Src/ERK/JNK/p38訊息傳導途徑活化，但沒有明顯影響cyclin D1、DMP1、p53、B-myb和Ets-1蛋白質合成。而這些現象於滲透壓控制組之高甘露糖(400 mg/dl)作用下並未被觀察到。另一方面細胞凋亡的現象似乎沒有在上述高葡萄糖的處理情況下被發現，因為Bcl-2與PARP蛋白質表現、caspase 3活性並無任何變化。
Probucol是一種雙酚化合物具有降血脂作用和抗氧化功效，可逆轉動脈粥狀硬化。因此，我們也分析抗氧化劑probucol對高葡萄糖誘發腎小管細胞過度肥大的影響。結果發現probucol、Src家族激酶抑制劑PP2和ERK激酶抑制劑PD98059都能顯著減弱高葡萄糖抑制細胞生長和高葡萄糖所誘發的Src、ERK訊息途徑活化以及細胞過度肥大。此外，在這些細胞經由probucol和N-acetylcystein處理後均可逆轉高葡萄糖所減少之抗氧化酶glutathione S-transferas活性。另一方面，probucol、PP2、PD98059也都能明顯抑制腎小管細胞過度肥大，因為它們可以阻斷高葡萄糖所誘發之第四型膠原蛋白、p27Kip1蛋白質的表現。因此從這些結果推測，probucol對高葡萄糖誘導腎小管上皮細胞過度肥大具有明顯的抑制作用，而Src、ERK信號傳導途徑可能是probucol重要的作用目標。
Diabetic nephropathy (DN) is the chief cause of new dialysis patients in our country. Most studies focused on the pathomechanisms of glomerular and tubulointerstitial cells in DN, which is characterized by cellular hypertrophy/hyperplasia and extracellular matrix expansion leading to renal fibrosis and end-stage renal disease. Two key mediators implicated in the development of DN include high glucose (HG) and reactive oxygen species. HG act to increase oxidative stress, promote extracellular matrix protein synthesis and tubulointerstitial fibrosis, and exert a number of toxic effects of renal cells. Renal tubular hypertrophy may be the first step in an inevitable pathophysiologic course, leading ultimately to tubulointerstitial fibrosis and diabetic nephropathy. However, the mechanisms of antioxidants on the HG-induced oxidative stress and renal tubular hypertrophy in DN remain unclear.
In this study, we examined the effect of HG on renal tubular growth and the role of Src/ERK signaling in the regulation of cellular hypertrophy. We found that HG inhibited cellular growth dose-dependently in human renal proximal tubular epithelial cells. In addition, HG (500 mg/dl) significantly enhanced the Src/ERK/JNK/p38 signaling activation but not cyclin D1, DMP1, p53, B-myb, and Ets-1 protein expression. These effects were not observed when cells were treated with the osmotic control high mannitol (400 mg/dl). It seems that apoptosis was not observed in these treatments. There were no changes in caspase 3 activity, Bcl-2 and poly(ADP-ribose) polymerase expression in HG-treated cells.
Probucol is a diphenolic compound with lipid-lowering effects and antioxidant properties that reverses atherosclerosis. Thus, the effect of probucol on HG-induced renal tubular hypertrophy was also investigated. We found that the antioxidant probucol, the Src family kinase inhibitor PP2, and the ERK inhibitor PD98059 treatments significantly attenuated HG-inhibited cellular growth and HG-induced the Src/ERK activation and cellular hypertrophy. Moreover, probucol and N-acetylcystein treatments reversed HG-reduced the antioxidizing enzyme glutathione S-transferase activity in these cells. The ability of probucol, PP2, or PD98059 to ameliorate renal tubular hypertrophy was also verified by the observation that it significantly blocked HG-increased the protein levels of collagen IV and p27Kip1. Hence, these results suggested that probucol has potent inhibitory effect against HG-induced renal tubular hypertrophy, and the Src/ERK signaling pathway may be important target of probucol