馬兜鈴酸 （Aristolochic acid） 是馬兜鈴科植物中特有的成分，過去研究顯示含馬兜鈴酸的中草藥會引發人體腎臟病變，但相關毒理研究多著重於成體毒性之探討。由於馬兜鈴酸的胚胎毒性及毒理機制目前仍不清楚，本研究以斑馬魚胚胎來探討馬兜鈴酸對胚胎發育之影響。首先，我們以馬兜鈴酸（10 ppm）浸泡處理24 hpf的斑馬魚胚胎7小時，發現馬兜鈴酸對48 hpf的胚胎腎臟 （原腎小管、原腎輸送管及主要影響部位為腎絲球） 及心臟 （構型異常、心包腔腫大和卵黃下方積血） 皆造成嚴重損傷，且72 hpf的胚胎存活率僅剩下14.2%。進一步觀測斑馬魚胚胎之腎絲球過濾率，發現浸泡馬兜鈴酸3小時後之斑馬魚腎功能剩下71.5±18.8%，而浸泡5小時後之腎功能僅有39.4±15.9%，表示馬兜鈴酸造成原腎損傷而導致腎臟功能喪失及存活率下降。此外，分析細胞死亡情形，馬兜鈴酸處理後會導致細胞凋亡（apoptosis）情況。觀察紅血球與白血球表現量，發現馬兜鈴酸處理後48hpf之斑馬魚，紅血球靜止不動及堆積在腎管及腸道附近，且紅、白血球數明顯變少。以 real-time PCR 分析馬兜鈴酸所引起之發炎反應，發現馬兜鈴酸會誘導 TNF-α、cox2及mpo 這些與發炎相關之基因表現量上升。在浸泡馬兜鈴酸之前以白藜蘆醇或熊果酸處理12小時，可抑制馬兜鈴酸所引發之發炎反應而減緩腎臟衰竭，但無法完全修復馬兜鈴酸造成的腎臟損傷。綜上所述，馬兜鈴酸毒性之細胞與分子機制可能是經由引發發炎反應造成腎臟損傷，引起造血功能異常，進而導致心臟及腎臟嚴重損傷，以致腎功能喪失，最終導致腎衰竭。

Aristolochic acid (AA) occurs naturally in Aristolochiaceae plants, and toxicological studies have shown the role of AA in human aristolochic acid nephropathy (AAN). Most studies have focused on the adult toxicity of AA, but the embryonic toxicity and toxicological mechanisms are still not clear. Therefore, we used zebrafish embryos to investigate the effects of AA on embryonic development and toxicity. After 24-hpf embryos were treated with AA (10 ppm) for 7 hours, the 48-hpf embryo kidney (glomeruli, pronephric tubules, and pronephric ducts) and heart (deformed heart, swollen pericardial cavity,and hemorrhagic yolk sac) were severely damaged, and the 72-hpf survival rate was decreased to 14.2%. The glomerular filtration rate (GFR) of embryos was further examined after AA treatment. The GFR of embryos after 3-h AA treatment decreased to 71.5 ± 18.8%, while that after 5-h AA treatment decreased to 39.4 ± 15.9%. Taken together, AA caused renal injury and dysfunction, as well as reduced survival rate of zebrafish embryos. Besides, AA-induced apoptosis was observed. A significant decrease in the amount of erythrocytes and leukocytes was observed in AA-treated embryos. Moreover, erythrocytes became stationary in blood flow and even accumulated in renal tubules and intestinal tract. Based on quantitative PCR, mRNA expression of inflammatory genes (TNF-α, cox2, and mpo) was upregulated in AA-treated embryos. 12-h treatment (12-24 hpf) of resveratrol or ursolic acid before AA treatment (24-31 hpf) was found to inhibit AA-induced inflammation and attenuate but not completely reverse AA-induced renal injury. In summary, the cellular and molecular mechanisms of AA toxicity may result in inflammatory renal injury and hematopoietic dysfunction, severe defects of heart and kidney, and ultimately lead to kidney dysfunction and failure.

中文摘要 ………………………………………………………………I
英文摘要 ……………………………………………………………III
目錄……………………………………………………………………IV
圖表目錄 ……………………………………………………………IX
縮寫表…………………………………………………………………XI

壹、前言…………………………………………………………………1
一、馬兜鈴酸其結構及特性…………………………………………2
二、馬兜鈴酸之分佈…………………………………………………2
三、馬兜鈴酸之代謝…………………………………………………3
四、馬兜鈴酸之藥理特性……………………………………………4
五、馬兜鈴酸所造成之腎臟病變……………………………………5
六、馬兜鈴酸之動物試驗……………………………………………8
七、馬兜鈴酸之毒理機制……………………………………………9
八、馬兜鈴酸之抗發炎反應機轉…………………………………10
九、熊果酸之相關資料……………………………………………11
十、白藜蘆醇之相關資料…………………………………………13
十一、斑馬魚的優勢………………………………………………14
十二、斑馬魚之腎臟發育…………………………………………15
十三、斑馬魚之心臟發育…………………………………………16
十四、本篇論文之動機與目的……………………………………17

貳、實驗材料與方法 ………………………………………………19
   一、實驗用斑馬魚( Danio rerio )之飼養 ………………………20
二、基因轉殖魚介紹………………………………………………20
三、斑馬魚胚胎收集………………………………………………21
四、馬兜鈴酸浸泡條件篩選………………………………………21
五、半數致死濃度( Median Lethal Concentration,LC50 ) ……21
六、免疫抗體螢光染色( Whole-mount immunostaining )………22
七、胚胎包埋及冷凍切片…………………………………………22
八、蘇木紫和伊紅染色法 ( Hematoxylin and Eosin Stain, H&E 
    Stain )…………………………………………………………23
九、TUNEL ( Terminal deoxynucleotidyl transferase-mediated dU
    TPbiotil nick end labeling ……………………………………23
十、YO-PRO and PI 染色法 ……………………………………25
十一、斑馬魚RNA萃取及cDNA合成…………………………25
十二、定量聚合酶鍊鎖反應(real-time quantitative PCR,Q-PCR).26
十三、探針( riboprobe )合成 ……………………………………27
十四、DNA接合反應 ……………………………………………27
十五、勝任細胞( competent cell )製備與大腸桿菌( Escherichia coli )轉型 ………………………………………………………27
十六、小量質體萃取………………………………………………28
十七、聚合酶鍊鎖反應( polymerase chain reaction,PCR ) ……29
十八、原位雜交法( whole-mount in situ hybridization )………29
十九、腎功能分析-腎絲球過濾率( Glomerular filtration rate, GFR )………………………………………………………31
二十、myeloperoxidase (MPO) staining …………………………31
二十一、Whole-mountο-dianisidine staining……………………32
二十二、設備………………………………………………………32

叁、實驗結果…………………………………………………………33
一、馬兜鈴酸的浸泡濃度，浸泡時間與導致腎臟之損傷情形成正比………………………………………………………………34
二、馬兜鈴酸對斑馬魚胚胎的半致死濃度(LC50)………………35
三、馬兜鈴酸造成斑馬魚腎臟之損傷……………………………36
四、原腎管細胞並未受到馬兜鈴酸影響而造成破裂……………37
五、馬兜鈴酸導致腎臟功能下降…………………………………37
六、馬兜鈴酸會引發急性腎衰竭…………………………………38
七、馬兜鈴酸引起急性發炎反應…………………………………38
八、馬兜鈴酸影響造血功能異常…………………………………39
九、馬兜鈴酸導致腎臟及心臟損傷………………………………40
十、白藜蘆醇及熊果酸可減緩馬兜鈴酸造成之腎臟損  傷 ……………………………………………………………41
十一、白藜蘆醇及熊果酸無法完全修復馬兜鈴酸造成之腎臟損傷 …………………………………………………………42

肆、討論………………………………………………………………44
一、不同時期之胚胎浸泡馬兜鈴酸所導致毒性影響之差異……45
二、馬兜鈴酸導致腎臟損傷其主要影響為腎絲球體 …………45
三、發炎反應誘發產生ROS，導致器官功能異常………………47
四、馬兜鈴酸會引起急性發炎反應，而導致腎臟衰竭 ………47
五、馬兜鈴酸導致斑馬魚造血功能之異常 ……………………48
六、馬兜鈴酸對於心臟的影響屬於晚期 ………………………50
七、紅血球浸潤導致器官異常…………………………………51
八、馬兜鈴酸對斑馬魚胚胎之毒理機制 ………………………52
九、白藜蘆醇和熊果酸減緩馬兜鈴酸所導致之發炎反應 ……………………………………………………………52
十、結論及未來展望 ……………………………………………54

伍、參考文獻…………………………………………………………55
陸、圖表………………………………………………………………65
柒、附錄………………………………………………………………90

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