研究cilostazol對於斑馬魚腎間腺組織發育的影響

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Title:	研究cilostazol對於斑馬魚腎間腺組織發育的影響
Other Titles:	Studying the effect of cilostazol on the development of zebrafish interrenal tissue
Authors:	陳予芙 Chen, Yu-Fu
Contributors:	劉薏雯 Liu, Yi-Wen 生命科學系
Keywords:	cilostazol;腎間腺組織;斑馬魚 cilostazol;interrenal tissue;zebrafish
Date:	2019
Issue Date:	2019-12-16T03:00:52Z (UTC)
Abstract:	Cilostazol為一種抗凝血劑，臨床上常用來治療週邊動脈堵塞。cilostazol已知可以透過抑制Phosphodiesterase III阻礙cAMP (Cyclic adenosine monophosphate) 的代謝使其在細胞內濃度上升。cAMP濃度上升會導致多項生理機制產生改變；其中已知Protein Kinase A (PKA) 被cAMP活化後會使StAR (steroidogenic acute regulatory protein, 類固醇生成速控蛋白) 基因表現之上升，在腎上腺細胞中影響類固醇生成 (steroidogenesis)，然而cilostazol是否影響腎上腺的發育和功能至今尚不清楚。我們因此想利用斑馬魚胚胎作為活體系統，探討cilostazol是否會影響類固醇生成以及類固醇生成組織的發育。實驗結果顯示，在不同濃度cilostazol處理下，斑馬魚胚胎的腎間腺組織大小隨cilostazol濃度上升而顯著增加。此外，在進行實驗時，斑馬魚幼魚的色素細胞與腎間腺組織染色後的位置緊密相鄰，使得腎間腺組織的觀察與定量較為費時，我們因此嘗試使用色素抑制劑1-phenyl 2-thiourea (PTU) 來解決此問題，但我們的實驗顯示cilostazol會與PTU產生藥物交互作用，以致影響cilostazol的藥性。因此我們繼而嘗試使用不同的透明斑馬魚品種來進行實驗，我們使用兩種透明魚種－golden與citrine來進行cilostazol的藥物處理實驗，結果顯示兩種透明魚種皆產生與野生種類似的cilostazol藥物處理效果，但golden與citrine 的體色透明度都無法達到便於我們觀察腎間腺組織的目的。然而。為了證明cilostazol的作用為藉由cAMP訊息傳導路徑，我們使用兩種已證實可增加cAMP濃度的藥劑－aminophylline與forskolin，來與cilostazol的藥效進行比較，我們發現非特異性抑制Phosphodiesterase作用的Aminophylline，在加藥實驗中對斑馬魚胚胎心跳和腎間腺組織的影響與cilostazol有相似的上升趨勢。另外，在forskolin的加藥實驗中，可以觀察到較高的胚胎毒性，與較深的色素表達。 Cilostazol is an anticoagulant, clinically used to treat peripheral arterial occlusion. cilostazol is known to increase the intracellular concentration of cAMP (cyclic adenosine monophosphate) , a second messenger in signaling mediated by G protein coupled receptor, by blocking its metabolism through the inhibition of phosphodiesterase III. Increasing the concentration of cAMP could cause changes in a multitude of physiological responses. Among them, it is known that the activation of protein kinase A (PKA) by cAMP increases the gene expression of StAR (steroidogenic acute regulatory protein) , which is crucial for steroidogenesis in the adrenal cells. However, whether and how cilostazol affects the development and function of the adrenal gland in vivo remains unclear. We therefore used zebrafish embryos to investigate whether cilostazol would affect steroidogenesis and the development of steroidogenic tissue. Our experimental results showed that the size of the interrenal tissue in zebrafish embryos was significantly increased at high concentrations (100 μM) of cilostazol treatment. Also, the cilostazol experiment performed in Tg(kdrl:GFP) fish was able to demonstrate a dose-dependent increase of steroidogenic activity at 5 dpf by the cilostazol treatment. In this experiment, the pigmentation of the wild type zebrafish tend to affect the observation and quantification of the interrenal tissue. When we tried to use the 1-phenyl 2-thiourea (PTU) to reduce the pigmentation, we found that PTU interfered with the pharmacological effect of cilostazol. We therefore tried to perform the cilostazol dosing experiment with two transparent fish lines, golden and citrine. The results of cilostazol treatments showed that both of the two transparent zebrafish strains demonstrated essentially similar phenotypes with those in wild-type zebrafish. However, neither golden nor citrine can improve the transparency for observing the interrenal tissue. In order to verify whether the cilostazol-caused phenotypes in the zebrafish embryo was due to the cAMP-mediated signaling pathway, we also studied the effects of two chemicals, aminophylline and forskolin, which increase intracullar cAMP concentrations through different mechanisms with those affected by cilostazol. We found that the non-selective phosphodiesterase inhibitor aminophylline led to a similar heartbeat phenotype as that in the cilostazol treatment. It remains to be studied whether cilostazol influences steroid production through the PKA-StAR pathway. It was also noted that forskolin treatments caused lethality and hyperpigmentation in zebrafish embryos.
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