跳到主要內容

臺灣博碩士論文加值系統

(18.97.14.86) 您好!臺灣時間:2025/02/20 05:57
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳鵬及
研究生(外文):Peng-Chi Chen
論文名稱:馬兜鈴酸誘導COX-2所牽涉之發炎反應會造成斑馬魚胚胎心臟衰竭
論文名稱(外文):Inflammation Involved COX-2 Induced by Aristolochic Acid Causes Heart Failure in Zebrafish Embryos
指導教授:游正博黃政鎮
指導教授(外文):John YuCheng-Chen Huang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:89
中文關鍵詞:馬兜鈴酸斑馬魚發炎反應心臟
外文關鍵詞:aristolochic acidzebrafishinflammationheart
相關次數:
  • 被引用被引用:0
  • 點閱點閱:361
  • 評分評分:
  • 下載下載:61
  • 收藏至我的研究室書目清單書目收藏:1
中草藥腎病變 ( Chinese herb nephropathy, CHN ) 的概念首先是在1992年被提出來,比利時 Vanherweghem 及其研究團隊指出有年輕婦女因為服用含有中草藥成份的減肥藥後,發生了急性腎間質纖維化的症狀。在進一步的植物生化分析,發現這些減肥藥都含有馬兜鈴酸的成份。Robisch 等人在 1982 年就已經證實馬兜鈴酸具有致突變性與致癌性。在一些哺乳動物實驗中,也已經證實馬兜鈴酸會直接影響到腎小管上皮細胞,尤其是近端腎小管,嚴重會導致腎小管壞死與腎間質纖維化。不過究竟馬兜鈴酸對腎臟所造成的影響是藉由什麼機制,是否會對其他器官造成毒性,或是影響胚胎的發育?所以我們利用斑馬魚系統,來了解馬兜鈴酸的毒性及上述問題。
當我們利用馬兜鈴酸去浸泡處理斑馬魚胚胎時,發現心臟出現嚴重缺陷。至於腎臟部份在外觀上則是沒有觀察到任何異常出現。在仔細觀察心臟的表現型,發現以下幾個缺陷:(1) 心臟變小且變形,(2) 心室的搏動能力幾乎喪失,(3) 心房的搏動能力嚴重下降,(4) 心臟內腔空間縮小,以及心臟功能喪失後,所併發的缺陷。例如:卵黃囊下方積血,圍心窩水腫,之後血液循環停止,胚胎死亡。這些心臟缺陷,後來我們認為是引起心臟衰竭。
為了瞭解馬兜鈴酸對斑馬魚胚胎真正作用的時期,分別對不同時期的胚胎進行藥物處理。對晚期胚胎進行藥物處理時,心臟所出現的缺陷與藥物浸泡處理之早期胚胎是相同的,所以我們認為馬兜鈴酸主要是影響心臟的功能。觀察馬兜鈴酸浸泡處理的胚胎心臟都可以順利發育至受精後 22 小時,甚至可以正常搏動。所以我們推測以馬兜鈴酸浸泡處理早期斑馬魚胚胎,胚胎會累積馬兜鈴酸,直到心臟發育完成,馬兜鈴酸才會開始影響心臟功能。為了了解馬兜鈴酸所影響的心臟搏動能力,我們假設馬兜鈴酸會影響心肌的結構。因此,我們利用 MF20 ( 染肌小節 ) 抗體免疫染色,發現馬兜鈴酸處理過的胚胎心肌有凝集 ( aggregation ) 的現象。為了瞭解心肌結構更細微的問題,利用穿透式電子顯微鏡 ( TEM ) 觀察,發現心室心肌纖維變少,剩餘的心肌纖維結構則是變的較為鬆散,甚至有崩解的情形。心房纖維則是也有排列不整齊或是崩解的情形。同時,我們也觀察心臟功能性基因 — amhc 與 cmlc2 的表現。但是,其維持心臟功能的重要基因 ( amhc 與 cmlc2 ) 的表現量則是沒有顯著的差異。所以,我們認為馬兜鈴酸主要是影響心肌肌纖維的結構。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 IL-1β、SAA、COX-2 表現量上升。當我們加入選擇性 COX-2 抑制子-NS-398,發現會抑制 COX-2 與 SAA 的表現,而減緩發生心臟衰竭的速度。
我們的研究同時也對馬兜鈴酸的劑量及藥性進行分析。找出馬兜鈴酸對斑馬魚胚胎心臟作用之半有效濃度 ( EC50 ) 為 5uM。我們也發現馬兜鈴酸的毒性是累積性的。例如:低劑量馬兜鈴酸浸泡處理的胚胎或是浸泡處理時間較短,都會使心臟缺陷的狀況延後出現。在10uM 馬兜鈴酸作用下,最早作用胚胎時間為 6hpf,而所需的最短處理時間為 4 小時。
我們的結果顯示馬兜鈴酸毒性的細胞與分子機制可能是經由引起發炎反應進而造成心臟衰竭。這些結果是在其他動物模式系統中所未被發現的。
“Chinese herbs nephropathy” (CHN) is a rapidly progressive nephropathy that was first described in 1992. Vanherweghem and his colleagues in Belgian found that women who had followed a particular weight-reducing regimen that included Chinese herbs developed CHN. Further plant biochemistry analyses revealed that the weight-reducing regimen contained the components of aristolochic acid (AA), which has been identified to be one of the causes to CHN. Robisch further showed that AA is also a mutagenic and carcinogenic agent. It has been reported that AA can cause tubular necrosis and interstitial renal fibrosis. However, the cellular or molecular mechanism of AA toxicity is largely unknown. We want to study the toxicity of AA using zebrafish as a model system.
When treated zebrafish embryos with AA, we did not observe any defect in zebrafish kidney. Surprisingly, we found severe defects in the hearts. The heart phenotypes include 1) small and deformed heart, 2) less contractibility in atrium, 3) loss of contractibility in ventricle, 4) small heart lumen, and the other complications such as blood pool under the yolk, pericardial edema, termination of blood circulation and lethality. The heart phenotypes of AA-treated embryos are highly penetrant and reproducible.
In order to distinguish whether AA affects the development or the physiology of the heart, we treated zebrafish embryos at different stages. We found that treatment on both young and old embryos causes similar phenotypes at later times. Also, we observed normal heart development in the AA-treated embryos up to at least 22 hours post fertilization when hearts begin to pump. These results suggest that AA may not directly affect heart development but its toxicity can be stored through embryogenesis and becomes affective after heart begins to function. To further understand the cellular defects underlie the contractibility phenotype, we tested whether AA affects the cardiac fiber structure by whole mount immunostaining with MF20 antibody which stains sarcomeres. AA treated hearts appear to have aggregation of cardiac fibers. By transmission electron microscopy, we observed dramatic defects in the cardiac fiber organization. The ventricular muscle fibers are largely missed and the remaining fibers appear loose and broken. Similarly, the atrial muscle fibers are disorganized and broken. However, we found that the expression of atrial muscle heavy chain (amhc) and cardiac muscle light chain 2 (cmlc2) is not significantly altered by AA treatment. Thus, we concluded that AA deleteriously affects the structure of cardiac fibers. By real-time PCR, we found that AA can induce the expression of IL-1β, SAA, COX-2 genes, which are involved in inflammatory response. To test whether inflammation is responsible for the AA toxicity, we treated zebrafish embryos with the anti-inflammation drug NS-398, which is a selective COX-2 inhibitor, together or after AA treatment. We found that NS-398 can attenuate the AA toxicity significantly. Furthermore, NS398 treatment reduces the expression level of COX-2 and SAA genes.
Our studies using zebrafish embryos also provide insights into the toxicological mechanisms of AA. We first found that the EC50 is 5uM and AA toxicity is accumulative. For example: lower concentration or shorter treatment of AA will take longer time to cause the heart defects. With 10uM AA, we found that the minimal AA treatment time to generate toxicity is 4 hours.
In summary, AA might trigger inflammatory response that causes specific destructive consequences to the cardiac muscle fibers. Our results with zebrafish embryos provide great details of cellular and molecular mechanisms of AA toxicity which is not yet possibly determined with other existing experimental systems.
中文摘要...............................................1
英文摘要 ( Abstruct )..................................4
壹、前言...............................................7
貳、實驗材料與方法....................................15
參、實驗結果..........................................40
肆、討論..............................................58
伍、參考文獻..........................................65
陸、圖表..............................................70
柒、藥品配方..........................................87
行政院衛生署。九十二年十一月四日署授藥字第0920002350 號公告。

行政院衛生署。藥物食品檢驗局。http://www.nlfd.gov.tw/aristo/臺灣馬兜鈴植物.doc

羅東博愛醫院。衛教資料:心血管手冊。

Adamopoulos, S., Parissis, J. T. and Kremastinos, D. T. (2001). A glossary of circulating cytokines in chronic heart failure. European Journal of Heart Failure 3, 517-526.

Aukrust, P., Ueland, T., Lien, E., Bendtzen, K., Muller, F., Andreassen, A. K., Nordoy, I., Aass, H., Espevik, T. and Simonsen, S. (1999). Cytokine network in congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. The American Journal of Cardiology 83, 376-382.

Beis, D., Bartman, T., Jin, S. W., Scott, I. C., D'Amico, L. A., Ober, E. A., Verkade, H., Frantsve, J., Field, H. A., Wehman, A. et al. (2005). Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development. Development 132, 4193-204.

But, P. P. and Ma, S. C. (1999). Chinese-herb nephropathy. Lancet 354, 1731-2.

Cheng, A. S., Chan, H. L., Leung, W. K., Wong, N., Johnson, P. J. and Sung, J. J. (2003). Specific COX-2 inhibitor, NS-398, suppresses cellular proliferation and induces apoptosis in human hepatocellular carcinoma cells. Int J Oncol 23, 113-9.

Cosyns, J. P., Goebbels, R. M., Liberton, V., Schmeiser, H. H., Bieler, C. A. and Bernard, A. M. (1998). Chinese herbs nephropathy-associated slimming regimen induces tumours in the forestomach but no interstitial nephropathy in rats. Arch Toxicol 72, 738-43.

De Broe, M. E. (1999). On a nephrotoxic and carcinogenic slimming regimen. Am J Kidney Dis 33, 1171-3.

Dinarello, C. A. (2000). Proinflammatory Cytokines. Chest 118, 503-508.

Drummond, I. A., Majumdar, A., Hentschel, H., Elger, M., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C. F., Stemple, D. L., Zwartkruis, F., Rangini, Z. et al. (1998). Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development 125, 4655-4667.

Ganshirt, H. (1953). [Isolation of aristolochic acid from various Aristolochiaceae and its quantitative determination.]. Pharmazie 8, 584-92.

Kanaan, N., Cosyns, J. P., Jadoul, M. and Goffin, E. (2003). The importance of a histology-based diagnosis of interstitial nephropathy in two patients with renal insufficiency. Nephrol Dial Transplant 18, 440-2.

Kelly, R. A. and Smith, T. W. (1997). Cytokines and cardiac contractile function. Circulation 95, 778-781.

Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of Embryonic-Development of the Zebrafish. Developmental Dynamics 203, 253-310.

Lin, B., Chen, S., Cao, Z., Lin, Y., Mo, D., Zhang, H., Gu, J., Dong, M., Liu, Z. and Xu, A. (2006). Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: Striking similarities and obvious differences with mammals. Molecular Immunology In Press, Corrected Proof.

Mann, D. L. and Young, J. B. (1994). Basic mechanisms in congestive heart failure. Recognizing the role of proinflammatory cytokines. Chest 105, 897-904.

Mengs, U. (1983). On the histopathogenesis of rat forestomach carcinoma caused by aristolochic acid. Arch Toxicol 52, 209-20.

Mengs, U. (1987). Acute toxicity of aristolochic acid in rodents. Arch Toxicol 59, 328-31.

Mengs, U. (1988). Tumour induction in mice following exposure to aristolochic acid. Arch Toxicol 61, 504-5.

Mengs, U., Lang, W. and Poch, J. A. (1982). The carcinogenic action of aristolochic acid in rats. Archives of Toxicology 51, 107-119.

Mengs, U. and Stotzem, C. D. (1993). Renal toxicity of aristolochic acid in rats as an example of nephrotoxicity testing in routine toxicology. Arch Toxicol 67, 307-11.

Mix, D. B., Guinaudeau, H. and Shamma, M. (1982). The Aristolochic Acids and Aristolactams. Journal of Natural Products 45, 657-666.

Pailer, M., Belohlav, L. and Simonitsch, E. (1955). Zur Konstitution der Aristolochiasauren. Monatshefte für Chemie / Chemical Monthly 86, 676-680.

Paulus, W. J. (1999). How are cytokines activated in heart failure? European Journal of Heart Failure 1, 309-312.

Pressley, M. E., Phelan Iii, P. E., Eckhard Witten, P., Mellon, M. T. and Kim, C. H. (2005). Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish. Developmental & Comparative Immunology 29, 501-513.

Raynes, J. G. and McAdam, K. (1991). Serum Amyloid-a Isoforms in Inflammation. Scandinavian Journal of Immunology 33, 657-666.

Saxen, L. and Sariola, H. (1987). Early Organogenesis of the Kidney. Pediatric Nephrology 1, 385-392.

Serluca, F. C. and Fishman, M. C. (2001). Pre-pattern in the pronephric kidney field of zebrafish. Development 128, 2233-41.

Stainier, D. Y. and Fishman, M. C. (1992). Patterning the zebrafish heart tube: acquisition of anteroposterior polarity. Dev Biol 153, 91-101.

Stainier, D. Y., Lee, R. K. and Fishman, M. C. (1993). Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development 119, 31-40.

Testa, M., Yeh, M., Lee, P., Fanelli, R., Loperfido, F., Berman, J. W. and Lejemtel, T. H. (1996). Circulating Levels of Cytokines and Their Endogenous Modulators in Patients With Mild to Severe Congestive Heart Failure Due to Coronary Artery Disease or Hypertension. Journal of the American College of Cardiology 28, 964-971.

Vanhaelen, M., Vanhaelen-Fastre, R., But, P. and Vanherweghem, J. L. (1994). Identification of aristolochic acid in Chinese herbs. Lancet 343, 174.

Vanherweghem, J. L., Depierreux, M., Tielemans, C., Abramowicz, D., Dratwa, M., Jadoul, M., Richard, C., Vandervelde, D., Verbeelen, D., Vanhaelen-Fastre, R. et al. (1993). Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 341, 387-91.

Yelon, D., Horne, S. A. and Stainier, D. Y. (1999). Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish. Dev Biol 214, 23-37.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top