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。

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.

目錄
謝誌……………………………………………………………………Ⅰ
中文摘要………………………………………………………………Ⅱ
英文摘要………………………………………………………………Ⅲ
目錄……………………………………………………………………Ⅴ
圖表目錄………………………………………………………………Ⅹ

第一章 前言	2
1-1查耳酮 (Chalcone ) 來源與衍生物	2
1-2 Chalcone與其衍生物之應用	4
1-3鈣離子不正常調控對細胞的影響	7
1-4模式物種─斑馬魚的優勢	8
1-5研究動機	9
第二章 材料與方法	10
2-1野生型斑馬魚（AB strain）的飼養	10
2-2斑馬魚胚胎收集	10
2-3聚合酶連鎖反應（Polymerase Chain Reaction, PCR）	11
2-4浸泡chalcone 衍生物	11
2-5原位雜交反應和合成RNA探針	12
2-6樣品包埋及冷凍切片（Embedding and Cryosection）	17
2-7蘇木紫與伊紅染色（H&E staining）	17
2-8斑馬魚胚胎核酸( Ribonucleotide acid, RNA ) 之萃取	18
2-9反轉錄聚合酶連鎖反應（Reverse transcription-PCR, RT-PCR）	18
2-10即時偵測同步定量聚合酶連鎖反應（quantitative real-time polymerase chain reaction, Q-PCR）	19
2-11螢光抗體染色法（Fluorescent Whole-mount immunostaining）	19
2-12吖啶橙染色（Acridine Orange staining）	20
2-13 YO-PRO-1與 PI染色（YO-PRO-1 Propidium Iodide staining）	21
2-14偵測活性氧分子（Reactive oxygen species, ROS）	21
2-15螢光顯微鏡、顯微照相系統	22
第三章 結果	23
3-1查耳酮 ( chalcone ) 衍生物之結構	23
3-2查耳酮衍生物之浸泡方式與存活率	24
3-3查耳酮衍生物造成肌肉損傷與畸形率	25
3-4查耳酮衍生物（3′-HC）造成肌肉萎縮	26
3-5 浸泡3′-HC導致肌肉之粒線體行自體吞噬作用	27
3-6 查耳酮衍生物促使體內ROS含量上升造成胚胎變異	28
3-7 浸泡3′-HC導致胚胎產生許多凋亡細胞	28
3-8 3′-HC對肌肉相關因子之影響	29
3-9 3′-HC可影響體內鈣離子的平衡	31
3-10 咖啡因可減緩3′-HC造成之肌肉萎縮	32
第四章 討論	34
4-1 查耳酮衍生物產生ROS導致粒線體損傷	34
4-2 查耳酮衍生物影響粒線體之功能導致肌肉萎縮	36
4-3 查耳酮衍生物藉由鈣離子造成之影響	37
4-4 結論與外來展望	38
第五章 前言	40
5-1 Ribonucleotide reductase之功能	40
5-2 RRM2在癌細胞中扮演的角色	42
5-3 Sonic hedgehog（shh）藉由Gli促進癌細胞生成	43
5-4 RRM2與Gli1之間的關係	46
5-5 模式物種─斑馬魚的優勢	47
5-6 研究動機	47
第六章 材料與方法	49
6-1 斑馬魚之實驗	49
6-2 勝任細胞（Competent cell）之備製	49
6-3 大腸桿菌轉型反應（Transformation）	49
6-4 小量質體DNA抽取 ( DNA抽取、電泳、剪切、純化及黏合)	50
6-5 質體建構	53
6-6 顯微注射（Microinjection）	54
6-7 人類表皮癌細胞（A431）之培養	54
6-8 計算活細胞數（Trypan blue exclusion assay）	55
6-9 細胞DNA轉染（Tranfection）	55
第七章 結果	57
7-1 rrm2之啟動子含有疑似Gli之結合位	57
7-2 疑似Gli結合位於斑馬魚胚胎中之表現位置	58
7-3 細胞受損時GliBS能提升rrm2進入細胞核之機率	59
第八章 討論	60
8-1 -486/ -1含有CCAAT box能增強rrm2之表現	60
8-2 斑馬魚組織中rrm2的表現	61
8-3 pEGFPm 2X（-243/-234）此質體具專一表現之能力	62
8-4 -486/-1具有幫助rrm2進入細胞核之能力	63
8-5 結論與未來展望	64
第九章 參考文獻	65
附錄	90
圖表目錄
圖A類黃酮屬於非酮羥基多酚化合物	2
圖B蘊含查耳酮之藥用植物與其結構式	3
圖C查耳酮衍生物之命名方式	23
圖D已知影響體內鈣離子物質之結構式	32
圖E Shh/Gli訊號傳遞路徑	45
圖F rrm2啟動子之簡圖	57
Fig.1 Chalcone與其衍生物之結構式	73
Fig.2浸泡方式與chalcones不同條件下之存活率	74
Fig.3 Chalcones造成肌纖維彎曲與畸形率統計	75
Fig.4 Chalcone衍生物（3′-HC）造成肌肉萎縮	76
Fig.5浸泡3′-HC讓粒線體行自體吞噬導致肌纖維不完整	77
Fig.6 Chalcones 造成胚胎內生性ROS表現上升	78
Fig.7 Chalcone衍生物（3′-HC）以細胞凋亡造成傷害	79
Fig.8 Chalcone衍生物（3′-HC）影響MyoD與α-actin 的mRNA表現	80
Fig.9咖啡因可減輕3′-HC造成之畸形率，而Amlodipine反之	81
Fig.10藉由咖啡因提升細胞內鈣離子濃度，減緩3′-HC造成之肌肉萎縮	82
Fig.11 rrm2啟動子中含有疑似Gli之結合位與質體建構圖	83
Fig.12 GliBS於斑馬魚胚胎中之表現位置	84
Fig.13 GliBS於斑馬魚胚胎中之表現位置	85
Fig.14含GliBS之啟動子能幫助rrm2修復受損細胞	86
Fig.15 rrm2啟動子之結合位比較圖	87
Table 1反轉錄聚合酶連鎖反應所用之引子	88
Table 2顯微注射質體之胚胎螢光統計表	89

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