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研究生:鍾森元
研究生(外文):Sen-yuan Chung
論文名稱:探討醌(Quinone)的衍生Akt標靶化合物對斑馬魚胚胎血管發育的影響
論文名稱(外文):The effect of Quinone derived Akt-targeting compound on vascular development in zebrafish
指導教授:吳長益
指導教授(外文):Chang-Yi Wu
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2015
畢業學年度:104
語文別:中文
論文頁數:77
中文關鍵詞:血管新生細胞凋亡斑馬魚Akt訊息傳導路徑Akt標靶藥物血管內皮生長因子
外文關鍵詞:vascular endothelial growth factor (VEGF)angiogenesisAkt signal pathwayapoptosisAkt-targeting compoundQuinonezebrafish
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醌(Quinone)是一類含有共軛環己二烯二酮(conjugated diene cyclohexene)或環己二烯二亞甲基(cyclohexadiene dione dimethylene)結構的有機化合物的總稱。過去研究指出醌具有生物、藥理活性,因此發展出許多衍生物在臨床上使用。
Akt又稱為Protein Kinase B(PKB),Akt訊息傳導路徑(Akt signal pathway)對細胞的增生、遷移及血管新生扮演很重要的角色。因此,抑制Akt訊息傳遞具有很大的潛力來治療相關疾病。例如癌症,腫瘤細胞的分裂生長需要大量養分,所以腫瘤細胞周圍會有大量血管形成,為腫瘤提供了優良的生長環境。在一些抗癌研究中指出,透過抑制Akt訊息傳導路徑讓腫瘤細胞凋亡,也能觀察到血管新生(angiogenesis)受到抑制。由於血管新生與腫瘤細胞存活有密切關係,只要有效抑制血管新生也能進一步抑制腫瘤細胞的發展。
我的研究正是以醌為結構的Akt標靶藥物(Akt-targeting compounds),來探討當抑制Akt訊息路徑之後對斑馬魚胚胎血管發育的影響。Akt標靶藥物購買於Enamine Ltd. 的化學資料庫(REAL DataBase),透過生物資訊學的方式先篩選出12個以醌為結構的Akt標靶藥物,其中挑出4個(X2、X5、X8、X9)做測試,再針對X8及X9進一步探討影響胚胎血管發育的分子機制。我利用斑馬魚當作模式生物,以Akt標靶藥物處理斑馬魚胚胎,從螢光基因轉殖斑馬魚的血管與內皮細胞顯示,發現Akt標靶藥物會造成血管內皮細胞數目減少,無法正常增生及遷移而導致血管發育異常。再利用全覆式原位組織染色(in-situ hybridization)檢測血管標誌基因的表現量,結果也出現下降趨勢,可見Akt標靶藥物對斑馬魚血管發育造成了不良影響。
當Akt傳導路徑受抑制,很可能導致血管內皮細胞進行細胞凋亡而減少,但是我們利用TUNEL assay及Acridine orange (AO) staining來檢測血管內皮細胞並無明顯細胞凋亡。通常血管新生的進行是受到血管內皮生長因子(VEGF)誘導,因此另外一種可能就是和VEGF有關。於是我們假設既然Akt標靶藥物會抑制斑馬魚血管新生的進行,VEGF的表現量應該會下降。進一步利用西方墨點法來檢測VEGF及其他與血管新生相關的蛋白質,如ERK、MMPs等,確認了這些Akt標靶藥物能透過抑制Akt訊息傳導路徑有效干擾血管新生。
Quinone (Quinone) is a general term for a class of organic compounds containing conjugated cyclohexadiene-dione (conjugated diene cyclohexene), or cyclohexadiene dimethylene (cyclohexadiene dione dimethylene) structure. Previous studies have pointed out that quinone has biological and pharmacological activity, thus many derivatives of quinone were developed for clinical use.
Akt is also called Protein Kinase B (PKB). Akt signal pathway plays an important role in cell proliferation, migration and angiogenesis. Thus, inhibition of Akt messaging has great potential to treat related diseases, such as cancers. Division and growth of tumor cells requires a lot of nutrients; therefore, stimulation of angiogenesis provides a good environment for tumor growth. It has been demonstrated that inhibition of Akt pathways resulted in apoptosis of tumor cells and suppression of angiogenesis. Since angiogenesis and tumor cell survival are closely related, as long as angiogenesis is effective inhibited, the development of tumor cells may also be diminished.
My study is to use quinone as the Akt-targeting compound to investigate the effects of Akt pathway inhibition on vascular development of zebrafish embryos. Akt targeting drugs was purchased from a chemical library Enamine Ltd. (REAL DataBase). We first used bioinformatics way to screen out 12 quinone structured Akt targeting drugs. Among which, four of them were tested (X2, X5, X8, X9) and X8 and X9 were selected for further investigation to determine the effects and molecular mechanism on embryonic vascular development. I use zebrafish as model organism to study Akt-targeting compound on zebrafish embryos. On vascular endothelial cells of fluorescent transgenic zebrafish, Akt-targeting compound treatment resulted in reduced number of vascular endothelial cells and decreased proliferation and migration of vascular endothelial cells which led to vascular abnormalities. In-situ hybridization was further used to detect the amount of gene expression of vascular markers. The results also show a downward trend, which indicates that targeting Akt had a negative impact on zebrafish vascular development
When Akt pathways were inhibited, it is likely to cause apoptosis of vascular endothelial cell and thus reduced cell numbers, but there is no significant difference in apoptosis of endothelial cells when we used TUNEL assay and Acridine orange (AO) staining to detect cell apoptosis. Angiogenesis is generally induced by vascular endothelial growth factor (VEGF), therefore VEGF may be related. We assume that since Akt targeting drugs can inhibit angiogenesis in zebrafish, the amount of VEGF expression should be decreased. Western blotting should be further used to detect the expression of VEGF and other angiogenesis-related proteins, such as ERK, MMPs, etc., to confirmed these drugs can effectively inhibits angiogenesis through the inhibition of Akt pathways.
論文審定書 i
誌謝 ii
摘要 iii
Abstract v
圖次 x
表次 xi
中英文名詞對照表 xii
Abbreviation xiii
第一章、前言 1
1.1 醌類化合物的特性 2
1.1.1 醌類化合物的病理研究 3
1.1.2 醌類結構的Akt標靶藥物 4
1.2 AKT訊息傳導路徑 5
1.2.1 Akt 家族 5
1.2.2 Akt 訊息傳導路徑與血管新生 6
1.3 RAS/RAF/MEK/ERK訊息傳導路徑與血管新生 7
1.4 SAPK/JNK訊息傳導路徑與血管新生 7
1.5 VEGF訊息傳導路徑與血管新生 7
1.6 MMPS與血管新生 8
1.7 模式生物斑馬魚的優點 9
1.8 斑馬魚胚胎的血管發育 9
1.8.1 斑馬魚胚胎血管的分化 9
1.8.2 脈管生成與血管新生 10
1.9 研究目的 11
第二章、材料與方法 12
2.1 實驗材料 12
2.1.1 使用的斑馬魚品系 12
2.1.2 Akt標靶化合物 12
2.2 實驗方法 12
2.2.1 斑馬魚的飼養與繁殖 12
2.2.2 斑馬魚胚胎的收集與培養 13
2.2.3 斑馬魚的藥物測試 13
2.2.4 斑馬魚total RNA 萃取(GeneMark Kit) 13
2.2.5 cDNA製備(Roche Kit) 14
2.2.6 Quantitative PCR (Q-PCR) 14
2.2.7 探針備製DIG RNA Labeling Probe (Roche kit) 14
2.2.8 全覆式原位組織染色(whole-mount in-situ hybridation) 15
2.2.9 Acridine orange (AO) staining 16
2.2.10 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay 16
2.2.11 斑馬魚蛋白質萃取 17
2.2.12 測蛋白質濃度 18
2.2.13 Western blotting assay 18
2.3 影像處理與數據統計分析 20
第三章、實驗結果 21
3.1 AKT標靶藥物的濃度測試 21
3.1.1 斑馬魚胚胎的存活率 21
3.1.2 Akt標靶藥物影響胚胎血管發育的情形 22
3.2 AKT標靶藥物影響斑馬魚胚胎血管生成的情形 23
3.2.1 胚胎發育的節間血管與尾部靜脈叢生長之差異 23
3.2.2 胚胎發育出現血液循環系統的缺陷 24
3.2.3 胚胎發育出現心包膜腫大的情形 25
3.3 AKT標靶藥物造成血管缺陷的分子機制 25
3.3.1 Akt標靶藥物並非造成血管內皮細胞凋亡 25
3.3.2 Akt標靶藥物造成血管內皮細胞數目的減少 26
3.4 AKT標靶藥物造成動、靜脈基因MARKER表現量下降 26
3.5 AKT標靶藥物干擾血管新生的訊息傳導 27
3.5.1 Akt標靶藥物抑制血管新生,與VEGF的減少有關 27
3.5.2 Akt標靶藥物抑制Akt磷酸化的程度,評估藥效 28
3.5.3 Akt標靶藥物抑制血管新生,可能有其他訊息路徑來回饋控制修補血管生長 28
第四章、討論 30
4.1 AKT標靶藥物劑量濃度高低對胚胎發育影響程度不同 30
4.2 AKT標靶藥物的設計理念 30
4.3 AKT標靶藥物抑制AKT磷酸化的效力 31
4.4 AKT訊息傳導路徑可能與P-AKT濃度高低進行選擇性調控下游目標 32
4.5 AKT標靶藥物抑制血管內皮細胞增生與遷移 33
4.6 AKT標靶藥物抑制AKT訊息路徑而降低了VEGF產生 33
4.7 WESTERN BLOTTING ASSAY用於斑馬魚蛋白質取樣的建議 34
4.8 AKT標靶藥物干擾NOTCH訊息路徑造成血管缺陷 35
參考文獻 54
附錄 59
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