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研究生:宋翊淳
研究生(外文):Yi-Chun Song
論文名稱:海洋天然物 GB9 對斑馬魚血管發育之影響
論文名稱(外文):Coral-derived natural marine compound GB9 inhibits 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:2017
畢業學年度:105
語文別:中文
論文頁數:66
中文關鍵詞:斑馬魚海洋天然物GB9血管新生
外文關鍵詞:zebrafishmarine compoundangiogenesisGB9
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血管發育對於脊椎動物極為重要,影響血管發育有許多因素,除了內在的基因表現及訊號調控以外,外在的因子,如小分子化合物、藥物、毒化物和環境賀爾蒙的刺激也有可能造成血管生缺陷或過度生長。已知海洋天然物GB9能減輕傷害性引起的疼痛和抗神經發炎的作用,但是對於血管的影響仍是未知,因此利用斑馬魚作為生物模式,探討GB9對血管發育的影響。實驗結果顯示,GB9處理斑馬魚胚胎會造成區間血管(ISV, intersegmental vessel)生長延遲,抑制尾部靜脈叢(CVP, caudal vein plexus)出芽生長,進而導致胚胎產生心包膜腫大現象和血液循環缺陷。由TUNEL assay 和AO staining得知,血管缺陷並不是由細胞凋亡所導致,而是因為細胞的增生/遷移受到影響,再利用細胞增生指標基因p-HH3表現,發現GB9處理胚胎後會降低p-HH3蛋白表現量,顯示GB9會影響細胞增生;測量斑馬魚ISV在24~28hpf的細胞移動距離和內皮細胞EA.hy926 wound healing assay結果得知,處理GB9會抑制細胞遷移。接下來,我們想進一步瞭解造成血管缺陷的分子機制,由in situ hybridization、qPCR和Western blot實驗發現,GB9會造成動靜脈基因ephrinb2、flt4、mrc1、flk、stabilin 的表現下降,也會減少VEGF和BMP下游訊號蛋白表現,顯示GB9會影響調控斑馬魚血管生長的分子訊號。另外,我們也探討GB9造成血管缺陷是否因為胚胎的氧化壓力上升,利用共同處理低劑量的GB9和 H2O2的胚胎中,觀察到尾部靜脈叢出芽生長缺陷的現象比單一處理GB9或H2O2更加嚴重,而抗氧化劑N-acetyl-cysteine (NAC)的添加,則會減輕因GB9造成血管缺陷的現象,顯示GB9處理之胚胎會增加氧化壓力,可能進而導致血管缺陷。綜合以上實驗結果顯示, GB9影響斑馬魚胚胎的血管發育,可能因為GB9處理胚胎後,導致其降低調控血管生長相關的VEGF和BMP訊息路徑,以及造成氧化壓力上升有關。
Vascular development is important for vertebrates, and genetic control vascular patterning has been intensively characterized. In addition, many studies have been show environmental hormones and chemical compounds affect vascular growth. Natural marine compound GB9 was isolated from the marine soft coral Capnella imbricate, the study show GB9 had anti-neuroinflammatory and anti-nociceptive effects. However, the effect of GB9 on blood vessels is still unknown. In this study, we use transgene zebrafish as an animal model to understand the effect of vascular development in GB9 treated embryo. The results showed that GB9 treated embryos impair ISV (intersegmental vessel) growth, and CVP (caudal vein plexus) sprouting, which leads to pericardial edema and circulation defects. TUNEL assay and AO staining showed that vascular defects were not caused by apoptosis, but likely due to the impairment of cell proliferation and/or migration. We further showed GB9 treatment reduce cell proliferation marker p-HH3 and protein level, by counting p-HH3 immunostaining cell and Western blotting. In addition, we quantitated the migration distance of ISV between 24hpf and 28hpf and perform wound healing assay in endothelial cells EA.hy926. We show that GB9 treatment inhibits cell migration. Next, we examined the molecular mechanism of vascular defects by in situ hybridization, qPCR and Western blot, the results showed GB9 treatment decreases the expression of arteriovenous markers, ephrinb2, flt4, mrc1, flk and stabilin;the protein levels related to VEGF, BMP signal pathways. Those results suggest that GB9 interfere vascular signaling regulation in zebrafish. Besides, we examine whether GB9 treatment cause vascular defect due to the increase of oxidative stress? We co-treated low-dose GB9 and H2O2, observed that defects in CVP formation was more severe than single treatment. Moreover, antioxidants Acetyl-cysteine (NAC) can rescue vascular defects in GB9 treated embryos. These data suggested that GB9 exposure causes vascular defects mediated by an increase in oxidative. Together our data show that GB9 treatment affect vascular development mediated by the interference of VEGF and BMP signals by the enhance of oxidative stress.
論文審定書i
誌謝 ii
中文摘要 iii
英文摘要 iv
圖次 ix
表次 x
縮寫 xi
前言 1
海洋天然物GB9 1
血管生成與發育 1
ROS與氧化壓力 2
模式生物-斑馬魚 3
斑馬魚血管發育 3
研究動機與目的 4
材料與方法 5
一、斑馬魚胚胎培養 5
二、GB9配製與胚胎處理 5
三、TUNEL assay 6
四、AO(Acridine Orange) staining 6
五、免疫螢光染色Immunofluorescent staining 6
六、探針製作 7
七、原位組織雜交染色in situ hybridization 7
八、Total RNA萃取 8
九、製作cDNA 9
十、即時聚合酶連鎖反應(Quantitative real time polymerase chain reaction) 9
十一、蛋白質萃取 10
十二、西方墨點法western blot 10
十三、細胞培養 11
十四、MTT assay 12
十五、Wound healing 12
十六、影像拍攝 13
十七、統計分析 13
實驗結果 14
一、GB9對斑馬魚的毒性 14
二、GB9造成斑馬魚血管發育缺陷 14
三、GB9處理的斑馬魚出現心包膜腫大與血液循環缺陷現象 15
四、GB9抑制斑馬魚區間血管內皮細胞的增生/遷移 16
五、GB9抑制細胞增生 17
六、GB9抑制細胞遷移 18
七、GB9 treatment會降低斑馬魚動靜脈基因marker的表現 19
八、GB9 treatment 降低VEGF和BMP下游訊號的蛋白量 19
九、利用GB9和H2O2共同處理斑馬魚胚胎,發現GB9會增加氧化壓力,導致斑馬魚胚胎血管缺陷 20
十、抗氧化劑NAC (N-acetyl-cysteine),能夠降低GB9增加氧化壓力,減少血管生長缺陷 20
十一、總結 21
問題與討論 22
圖 25
表 38
參考文獻 40
附錄 44
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