第一部分：查耳酮衍生物對斑馬魚胚胎之安全評估；第二部分：rrm2基因之轉錄調控

PartI: Zebrafish as a model for safety assessments of chalcone derivatives.; PartⅡ: Transcriptional regulation of the rrm2 gene.

李雅婷(Ya-Ting Lee)

指導教授 ： 陳曜鴻

淡江大學/理學院/化學學系碩士班/碩士(2012年)

https://doi.org/10.6846/TKU.2012.00052

摘要

第一部分：查耳酮與其衍生物皆被認為具有許多藥理活性，包括抗發炎、抗癌及抗氧化。本篇論文利用發育中之斑馬魚胚胎來評估羥基查耳酮之安全性。我們的實驗結果顯示具羥基之查耳酮衍生物會造成胚胎軀幹彎曲、肌纖維排列不整齊等異常的表現型，其中以3′-hydroxychalcone（3′-HC）最為嚴重。結果顯示浸泡3′-HC會導致肌肉萎縮、促使骨骼肌之粒線體行自噬凋亡作用並且提升體內活性氧分子（ROS）的含量，利用TUNEL分析法也顯示3′-HC會造成細胞凋亡。若將3′-HC與可降低ROS含量的咖啡因同時浸泡可減輕胚胎畸形率，但鈣離子抑制劑-脈優則會加強其變異，最後利用即時定量RT-PCR得知咖啡因可降低3′-HC提升的肌肉萎縮相關基因Fbox32之表現。因此我們推論具羥基查耳酮能提升體內ROS含量，並使粒線體功能受損導致其肌肉萎縮且此變異與鈣離子調控有關。雖然in vivo實驗結果和細胞上有所差別，但未來或許能藉由浸泡胚胎之方式來研發更具有優異生物活性且低毒性之查耳酮衍生物。第二部分：核糖核酸還原酶（ribonucleotide reductase, RNR）之活性取決於細胞週期中M2次單元體的合成與降解且腫瘤細胞中皆能發現RNR酶有異常之活性。我們先前發表之論文證明，過度表現斑馬魚sonic hedgehog（shh）基因其內生性的gli1與rrm2的表現量也會跟著上升。經比對發現rrm2啟動子上-243/ -234之位置含有疑似Gli的結合位（GliBS），因此利用顯微注射斑馬魚胚胎探討此GliBS對於調控rrm2的重要性，啟動子 -486/ -1主要表現於胚胎肌肉與心臟，且含有GliBS之質體其螢光表現明顯強於只含TATA box（-222/-1）。接著利用含CMV TATA box之pEGFPm質體，得知似乎有轉錄因子能結合在GliBS（-243/-234）並且具有專一表現於肌肉之能力，而細胞實驗中也顯示-486/-222片段似乎具幫助rrm2進入細胞核之能力。未來或許能藉由-486/-222片段來影響rrm2之轉錄並降低RNR酶活性以加強抗癌療效，使癌細胞不易轉移並降低產生抗藥性機率。

關鍵字

斑馬魚 ； 查耳酮 ； 骨骼肌 ； 核糖核酸還原酶

並列摘要

PARTI:It was reported that chalcone and its derivatives possess various biological activities, including anti-inflammatory, anti-cancer and anti-oxidant properties. Aim of this study was to investigate the toxic effects of hydroxychalcones during zebrafish embryogenesis. After hydroxychalcones treatment [especially 3′-hydroxychalcone (3′-HC)], zebrafish embryos displayed deformed somite phenotypes, such as curved body and muscle fiber mis-alignment. Interestingly, those malformed phenotypes can be rescued by adding caffeine, but can be enhanced by adding amlodipine. To further investigate the cause of 3′-HC-induced deformed somite phenotypes, we carried out histocytochemistry and electron microscopy experiments. Results demonstrate that 3′-HC was able to induce muscle atrophy, mitochondrial autophagy and increased reactive oxygen species (ROS) levels. Furthermore, the increase in TUNEL-positive cells was only observed in those which were exposed 3′-HC. Finally, the reverse transcription- PCR analysis showed that caffeine can inhibit the up regulation of expression of Fbxo32 by 3′-HC. In conclusion, we suggested that 3′-HC induces apoptosis in muscle by alteration of mitochondrial calcium signalling and generation of ROS. PARTⅡ:The activity of ribonucleotide reductase M2 subunit (Rrm2) was reported to be highly associated with the tumorgenesity in a variety of mouse and human cells. We previously demonstrated that overexpression of sonic hedgehog (Shh) in a zebrafish model leads to upregulation of rrm2. Aim of this study was to investigate whether Gli (key regulator of Shh signaling pathway) is a direct upstream regulator of rrm2 gene or not. After sequence analysis, we found that there is only one Gli-binding site (positions -243 to -234, GliBS) locates within the proximal rrm2 promoter. In this regards, two GFP-expression plasmids, pRrm2(-486/-1)-GFP and pRrm2(-222/-1)-GFP, were constructed for promoter analysis. Microinjection data showed that pRrm2(-486/-1)-GFP and pRrm2(-222/-1)-GFP-injected embryos have green fluorescent signals on muscle and heart, but the pRrm2(-486/-1)-GFP-injected embryos have higher GFP-expression rates. Furthermore, cassette -243/-234 is able to direct muscle-specific expression of the cytomegalovirus (CMV) basal promoter. On the basis of these observations, we conclude that cassette -243/-234 might be a key regulator to drive muscle-specific rrm2 expression.

並列關鍵字

zebrafish ； chalcone ； skeletal muscle ； ribonucleotide reductase

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