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    Title: 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
    Other Titles: 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
    Authors: 林伯儒;Lin, Po-Ju
    Contributors: 淡江大學化學學系碩士班
    陳曜鴻;Chen, Yau-Hung
    Keywords: 急性腎衰竭;芫荽;馬兜鈴酸;腎臟;斑馬魚;奈米金桿;acute renal failure;Coriandrum sativum;aristolochic acid;kidney;Zebrafish;Gold Nanorod
    Date: 2014
    Issue Date: 2015-05-04 09:48:01 (UTC+8)
    Abstract: 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。
    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.
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