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系統識別號 U0002-1907201400134600
中文論文名稱 第一部分:芫荽水萃物對馬兜鈴酸腎毒性之減毒功效評估 第二部分:應用奈米金桿誘導斑馬魚螢光表現之評估
英文論文名稱 Part I:Nephroprotective role of Coriandrum sativum in aristolochic acid intoxicated zebrafish Part II:The assessment of the utility of GNR to induce expression of fluorescence in zebrafish larva
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 102
學期 2
出版年 103
研究生中文姓名 林伯儒
研究生英文姓名 Po-Ju Lin
電子信箱 bolu0401@gmail.com
學號 601180051
學位類別 碩士
語文別 中文
口試日期 2014-05-29
論文頁數 113頁
口試委員 指導教授-陳曜鴻
委員-董崇民
委員-蔡振寧
委員-陳曜鴻
中文關鍵字 急性腎衰竭  芫荽  馬兜鈴酸  腎臟  斑馬魚  奈米金桿 
英文關鍵字 acute renal failure  Coriandrum sativum  aristolochic acid  kidney  zebrafish  gold nanorod 
學科別分類 學科別自然科學化學
中文摘要 第一部分:以馬兜鈴酸 (AA)作為腎衰竭的致病因子,運用斑馬魚動物模式並藉由芫荽水萃物特性,減緩馬兜鈴酸引發的腎損傷,並利用腎臟細胞表現綠螢光蛋白的基因轉殖魚Tg(wt1b:GFP)作為模式物種評估芫荽對於馬兜鈴酸腎毒性的減毒功效。我們以0~20000 pmm濃度的芫荽水萃物對12 hpf的斑馬魚胚胎浸泡12小時,接著於24 hpf時浸泡3 ppm AA 7小時,17小時過後於48 hpf統計其存活率約在91 ~99% 之間。遵循上述浸泡流程,將腎絲球、腎小管、腎輸送管分別做評分統計,統計腎臟損傷情形。結果顯示僅在3 ppm AA 7小時浸泡下的斑馬魚胚胎腎臟畸形分數為1.67,我們取得最佳防護濃度為浸泡 100 ppm芫荽水萃物12小時以及3 ppm AA,畸形分數為0.77。接著在腎絲球過濾率分析之下,3~4 dpf的斑馬魚胚胎,在100 ppm 芫荽水萃物的防護之下腎功能提升約12%。接著利用定量即時聚合酶鏈鎖反應針對發炎指標基因TNF- α, mpo及細胞凋亡指標基因bax 作相對定量分析,在100 ppm 芫荽水萃物的防護之下TNF- α, mpo, bax的表現量分別是對照組(僅浸泡3 ppm AA)的0.271、0.604、0.678倍。藉由紅血球染色以及切片的觀察,發現血球淤積情形程度下降。綜合所有結果,我們推測芫荽對腎毒性的減毒功效可能是透過抑制NF-κB的表現,降低了發炎以及細胞凋亡的發生,減緩了腎臟的損傷程度。

第二部分:利用斑馬魚作為模式物種,評估奈米金桿之應用。以晶種生成法製備高產率的奈米金桿,製備奈米金桿可得到表面電漿共振頻率 (SPL,max)為801 nm的金桿溶液,藉由穿透式電子顯微鏡量測其長寬分別為36.08(±5.68) nm和9.55(±1.05) nm,長寬比值(AR)為4.01之奈米金桿。以1000 mW近紅外光照射15分鐘後,溫度可從25℃上升至50℃。我們透過顯微注射使奈米金桿進入斑馬魚1 cell胚胎中測試奈米金桿在胚胎的毒性。24 hpf 的斑馬魚存活率約85.7% (409/477),至72 hpf時仍有81.1% (387/477)。接著將帶有奈米金桿的斑馬魚胚胎於24hpf時,以1000 mW近紅外光照射5小時,72 hpf時的存活率仍有82.9% (63/76)。過顯微注射使zhp70p-EGFP基因轉殖進入斑馬魚1 cell胚胎中,24 hpf在40℃的環境下生長5小時並觀察螢光表現,結果顯示約有59.2% (170/287)的斑馬魚胚胎出現螢光表現,另一方面我們將斑馬魚胚胎1 cell 將zhp70p-EGFP及奈米金桿混合溶液透過顯微注射進入斑馬魚體內。接著遵循上述近紅外光照射之方法,我們將斑馬魚胚胎1 cell 將zhp70p-EGFP及奈米金桿混合溶液透過顯微注射進入斑馬魚體內,僅有29.8% (31/104)的斑馬魚胚胎出現螢光表現。根據結果,我們認為奈米金桿能夠應用在斑馬魚胚胎上,並且能藉由表面電漿共振效應行光熱轉換。隨著奈米金桿在斑馬魚模式物種的開發,未來我們希望能夠利用奈米金桿與其他奈米材料合成的複合材料應用在斑馬魚上,進行標靶腫瘤治療或是建立動物的心血管疾病模式。
英文摘要 Part I : In this study , the Aristolochic Acids (AA), is used as the pathogenic factor of acute renal failure. In the zebrafish animal model, the water extract of Coriander Sativum acts as a key factor to alleviate the acute renal failure caused by AA. The transgenic line, Tg(wt1b:GFP), is the animal model, and the green fluorescent protein (GFP), expressed from its kidney cells is the way to evaluate the effectiveness of the AA intoxicated attributed to the Coriander Sativum. The differnet concnetrations of Coriander Sativum water extract, from 0 ppm to 20000 ppm, were used for treating the stage of 12hpf zebrafish embryos under 12 hours. Following, the stage of 24 hpf of zebrafish embryo were treated for 3ppm of AA under 7 hours. After 17 hours, the survival rates of the stage of 48 hpf zebrafish embryo was around 91% to 99%. After the completion of the process above, the acute renal failure statistics was gathered by scoring the glomerular, pronephric ducts, pronephric tubules. Continuously, the RT- qPCR was used as the quantitative analysis of the gene of inflammatory marker, TNF- α, mpo, and the gene of cell apoptosis marker, bax.Under the nephroprotection of 100 ppm Coriander Sativum water extract,the quantity performances of TNF- α, mpo, and bax are 0.271, 0.604, and 0.678 times those of the control group in which the zebrafish embryo is treated for 3ppm of AA under 7 hours. Observing the RBC stain and the tissue sections, we found the degree of the accumulation of red blood cells obviously decreases. In summary, we speculate that the nephroprotection attributed to Coriander Sativumm is caused by inhibiting the performance of NF-κB, which decreases the inflammation, cell apotosis , and alleviates the acute renal failure.

Part II: In this study, we assess the utility of gold nonarod (GNRs) by zebrafish embryo. Uniform size of GNRs with a dimension of 36.08 (±5.68) nm in length and 9.55 (±1.05) nm in width resulting in an aspect rato (AR) of 4.01 and Longitudinal Surface plasmon resonance (SPL,max) at 801 nm were synthesized by seed-mediate growth method. After 15 minutes of 1000 mW NIR irradiation, the temperature of the GNR solution increased from 25℃ to 50℃ due to the surface plasmon resonance effect of the GNRs.We injected the GNRs to the stage of 1 cell embryo by micro injection for testing the GNR of embryo toxicity.The survival rate of the stage of 24 hpf zebrafish embryo was 85.7% (409/477) and that of the stage of 72 hpf zebrafish embryo was 81.1% (387/477). After 5 hours of 1000 mW NIR irradiation, the survival rate of the stage of 24 hpf zebrafish embryo which was injected in GNRs was 82.9% (63/76).The stage of 1 cell embryo was injected in the heat shock protein gene zhp70p-EGFP by the micro injection. The stage of 24 hpf zebrafish embryo was exposed to the environment with 40℃ under 5 hours and was observed with the expression of fluorescence. Consequently, there was around 59.2% of the zebrafish embryo with the expression of fluorescence.After that, the stage of 1 cell embryo was injected in the mix solution combined with zhp70p-EGFP and the GNR by the micro injection. Consequently, there was only 29.8% (31/104) zebrafish embryos with the expression of fluorescence.
論文目次 目錄
中文摘要: I
ABSTRACT III
目錄 VI
圖表目錄 XI
附錄目錄 XIII
第一部分:芫荽水萃物對馬兜鈴酸腎毒性之減毒功效評估 1
第一章 前言 2
一、腎臟之基本生理功能 2
二、急性腎衰竭(ACUTE RENAL FAILURE)之病因及根治 2
三、芫荽其成分及特性 7
四、芫荽具抗氧化效果並清除自由基 8
五、芫荽具有利尿效果 9
六、馬兜鈴酸特性概述 9
七、馬兜鈴酸腎毒性 10
八、馬兜鈴酸對斑馬魚胚胎的影響 11
九、斑馬魚的優勢 12
十、斑馬魚腎臟發育 13
十一、本篇論文之研究動機 14
第二章 實驗材料與方法 15
一、實驗用斑馬魚(DANIO RERIO)之飼養 15
二、斑馬魚胚胎收集 15
三、基因轉殖魚介紹 16
四、芫荽水萃取方法 16
五、毒素曝露及天然物防護 16
六、觀察腎臟發育及統計畸形率 17
七、腎功能分析-腎絲球過濾率(GLOMERILAR FILTRATION RATE, GFR) 18
八、紅血球染色(WHOLE-MOUNT O –DIANISIDINE STAIN) 19
九、包埋及冷凍切片( EMBEDDING AND CRYOSECTION) 19
十、斑馬魚胚胎RNA萃取 20
十一、反轉錄聚合酶連鎖反應 ( REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION, RT-PCR ) 21
十二、即時偵測同步定量聚合酶連鎖反應( QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION, QPCR ) 21
十三、儀器設備 22
第三章 實驗結果 23
一、芫荽的浸泡濃度不會影響斑馬魚的存活率 23
二、低濃度芫荽水萃物會減緩馬兜鈴酸對斑馬魚造成之腎毒性 24
三、芫荽水萃物會使腎損傷的斑馬魚腎功能提升 27
四、芫荽水萃物會降低腎損傷引起的發炎反應 28
五、芫荽水萃物會減緩腎損傷導致的造血功能異常 29
第四章 討論 31
一、芫荽水萃取物對於馬兜鈴酸腎毒有顯著的減毒功效 31
二、芫荽的利尿效果降低馬兜鈴酸腎毒性 32
三、芫荽的酚類成分有助於減緩腎臟受到損傷 33
四、芫荽對腎毒的減毒功效可能與NF –ΚB的抑制有關 34
第二部分:應用奈米金桿誘導斑馬魚螢光表現之評估 36
第五章 前言 37
一、奈米材料特性 37
二、奈米金桿 (GOLD NANOROD, GNR) 39
三、奈米金桿的製備 40
四、奈米金桿的穩定性 42
五、奈米金桿的光熱性質 43
六、奈米金桿於生物上之應用 44
七、斑馬魚優勢 47
八、研究動機 49
第六章 實驗材料與方法 51
一、奈米金桿 (GNR) 之製備 51
二、奈米金桿吸收峰測定(紫外光-可見光光譜分析,UV-VISIBLE SPECTROPHOTOMETRY) 53
三、奈米金桿(GNR) 形態分析(穿透式電子顯微鏡分析,TEM ) 53
四、奈米金桿質量測定 53
五、奈米金桿對激發光源之光熱效應 54
六、實驗用斑馬魚 ( DANIO RERIO )之飼養及胚胎收集 55
七、勝任細胞 (COMPENTMENT CELL)製備 55
八、大腸桿菌轉型反應 ( TRANSFORMATION) 55
九、小量質體DNA 抽取 56
十、斑馬魚胚胎顯微注射 57
十一、誘導熱休克蛋白質 ( ZHS70P –EGFP ) 之表現 59
十二、儀器設備 61
第七章 實驗結果 64
一、奈米金桿的性質與形態 64
二、奈米金桿的產量及濃度計算 65
三、奈米金桿對激發光源之光熱效應溫度測試 65
四、奈米金桿在斑馬魚胚胎毒性存活率分析 66
五、奈米金桿之光熱效應誘導熱休克蛋白 (ZH70P:EGFP) 基因螢光表現分析 68
第八章 討論 70
一、奈米金桿在斑馬魚動物模式下有良好的生物相容性 70
二、奈米金桿在斑馬魚體內由NIR長時間照射,能誘導熱休克蛋白螢光表現 71
三、奈米金桿複合材料在斑馬魚動物模式應用之未來展望 72
參考文獻 74
圖表 87
附錄 107



圖表目錄
圖1-1. 馬兜鈴酸 I (Aristolochic scid I)與馬兜鈴酸II (Aristolochic scid II)化學結構 10
圖2-1. 斑馬魚胚胎在不同時期浸泡芫荽水萃物及馬兜鈴酸溶液 17
圖6-1. 奈米金桿(GNR)製備流程圖 52
圖6-2. 斑馬魚透過NIR熱誘導實體裝置圖 60
Fig. 1-1. 斑馬魚胚胎浸泡0 ~20000 ppm芫荽水萃物之胚胎存活率 87
Fig. 1-2. 斑馬魚胚胎浸泡0 ~20000 ppm芫荽水萃物及馬兜鈴酸溶液之胚胎存活率 98
Fig. 1-3. 斑馬魚胚胎浸泡0 ~20000 ppm芫荽水萃物及馬兜鈴酸溶液
    之胚胎存活率比較 89
Fig. 2. 浸泡芫荽及馬兜鈴酸後導致斑馬魚腎臟畸形 90
Fig. 3-1a. 斑馬魚胚胎浸泡0 ~333 ppm芫荽水萃物及馬兜鈴酸溶液之
    腎臟畸形率 92
Fig. 3-1b. 斑馬魚胚胎浸泡400 ~20000 ppm芫荽水萃物及馬兜鈴酸溶液之腎臟畸形率 93
Fig. 3-2a. 斑馬魚胚胎浸泡0 ~400 ppm芫荽水萃物及馬兜鈴酸溶液之
    腎臟畸形分數 94
Fig. 3-2b. 斑馬魚胚胎浸泡400 ~20000 ppm芫荽水萃物及馬兜鈴酸溶
    液之腎臟畸形分數 95
Fig. 3-3. 浸泡0 ~20000 ppm芫荽及3 ppm馬兜鈴酸,腎臟損傷程度之比較 96
Fig. 4. 芫荽減緩馬兜鈴酸腎毒之腎絲球過濾率(GFR) 97
Fig. 5. 相對定量聚合酶連鎖反應觀察發炎指標基因(TNF α and mpo)
    及細胞凋亡(bax)之表現量 99
Fig. 6. 以紅血球染色 ( o –dianisidine staining )觀察芫荽及馬兜鈴酸浸泡處理後48 hpf 紅血球堆積情形 100
Fig. 7. GNR溶液以UV-Visible spectrophotometry 及TEM 形態分
析 102
Fig.8. GNR溶液經NIR持續照射30P 分鐘後溫度上升情形 103
Fig.9. 斑馬魚胚胎經GNR溶液顯微注射後存活率分析 104
Fig.10. 斑馬魚胚胎經熱誘導後,螢光表現面積範圍之評估 105
Fig.11. 斑馬魚胚胎24hpf時,經NIR照射後螢光表現之分析 106


附錄目錄
附錄一. 斑馬魚0~72 hpf之發育週期 107
附錄二. 斑馬魚腎臟發育過程 111
附錄三. zhs70p-EGFP質體DNA 構築圖 112
附錄四. 反轉錄聚合酶連鎖反應所用之引子 113
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