Thioacetamide (TAA) 是一種肝毒性物質，已證實處理老鼠和斑馬魚後均有肝癌的形成，但是對於動物胚胎發育是否具有毒性並不清楚。在先前的研究中，我們利用0-32 mM TAA處理後第六小時，胚胎的存活率顯著下降至約80%，並且心包膜有edema的現象，為了探討TAA造成edema的分子機制為何和基因表現的情形，我們利用組織切片分析斑馬魚胚胎發育時之細胞變化結果發現TAA造成 pericardial附近聚集許多已活化的Mast Cell，進而利用Microarray 分析與TAA抑制心臟早期發育相關基因nrp2b及mef2cb 的表現。利用RT-PCR和real-time qPCR定量的結果證實TAA處理後第6-24 hpf胚胎內mef2cb 和 nrp2b基因表現量減少一倍，接著利用mopholino去konck-down 胚胎內mef2cb 和 nrp2b基因的表現，明顯在24小時有56% 和55%心包膜出現edema，證實TAA可透過抑制nrp2b &;mef2cb基因表現而造成edema。本實驗室先前已經證明TAA可誘導胚胎大量產生H2O2 ，因此我們利用4 mM H2O2 處理胚胎6-24小時後，再以RT-PCR和real-time qPCR定量，發現胚胎內mef2cb 和 nrp2b基因表現量顯著減少2倍以上也造成胚胎edema顯著增加，存活率下降。我們也將胚胎處理4mM H2O2至60 phf後不僅會造成edema in pericardial sac也會使Mast Cell聚集在pericardial附近。由以上的結果可以得知TAA會誘導胚胎大量產生H2O2使nrp2b和mef2cb表現量下降並導致胚胎時期心包膜出現edema最終造成死亡。未來將證明Mast Cell的活化是否造成edema in pericardial sac主要因素。

Thioacetamide (TAA) is a hepatotoxin that can cause hepatocellular carcinoma in mice, but little is known about its toxic effect on embryonic development. In the previous study, we treated zebrafish embryos with 0-32 mM TAA for 0-72 h, the survival rate were decreased to about 80% and the edema in pericardial sac (eps) were shown. For understanding the mechanism about TAA teratogenesis, we used DNA microarray to analyze the gene expression in zebrafish embryos when responses to TAA treatment. After data mining, there are 46 genes associated with organogenesis, and 107 genes involved in regulation of transcription. Among these genes, nrp2b and mef2cb were down-regulated to 2 or 1.8 fold after TAA treatment. nrp2b is a receptor that VEGF-mediated vascular development and mef2cb as a novel regulator in heart development. We then test the TAA downregulated nrp2b and mef2cb that can cause the edema in pericardial sac. 32 mM TAA treated embryos showed significantly decreased nrp2b and mef2cb gene expression, knock-down either nrp2b or mef2cb caused 55% and 56% eps on 24hpf, the results were the same as TAA treated embryos. Our laboratory has demonstrated previously the TAA induce embryonic a lot of produce H2O2. Therefore, we use 4 mM H2O2. treatment embryonic 6-24 hours then of quantitative RT-PCR and real-time qPCR, significantly reduce the more than 2 fold also caused a increase in embryonic eps the embryo mef2cb nrp2b amount of gene expression and increased mortality rate. Embryos processing 4mM H2O2 to 60 phf will not only cause eps makes Mast Cell aggregation in the pericardial sac near。By the above results, you can see that the TAA induce embryonic large number of generated the H2O2 that cuase reduce nrp2b and mef2cb gene expression and lead to eps eventually leading to death.

目錄
中文摘要 II
ENGLISH ABSTRACT IV
誌謝 VI
縮寫表 VII
目錄 VIII
第一章、前言 1
第一節、肝毒性物質TAA 1
第二節、TAA造成EDEMA IN PERICARDIAL SAC (EPS)與細胞的關係 3
第三節、TAA造成EPS與基因的關係 5
第四節、ROS當作細胞生理的訊息分子 8
第五節、實驗動機 11
第二章、實驗材料 13
第三章、實驗步驟 19
第一節、TAA處理斑馬魚胚胎 19
第二節、TAA處理BMP4：EGFP基因轉殖魚胚胎 19
第三節、組織切片 19
第四節、MAST CELL 抑制劑 CROMOLYN與TAA 處理 23
第五節、MICROARRAY的作法 23
第六節、REVERSE TRANSCRIPTASE PCR ( RT-PCR ) 25
第七節、REAL TIME PCR 26
第八節、WHOLE MOUNT IN SITU HYBRIDIZATION 28
第九節、KNOCKDOWN 35
第十節、DCFH-DA ASSAY 37
第四章 結果 38
第一節、以肝毒性物質TAA處理WLID TYPE斑馬魚胚胎 38
第二節 TAA造成EDEMA IN PERICARDIAL SAC是經由MAST CELL活化
引起的 38
第三節、TAA抑制MEF2CB及NRP2B基因表現與EPS 形成的關聯性探討 42
第四節、利用DCFH-DA偵測TAA誘導胚胎內HYDROGEN PEROXIDE 44
第五節、H2O2 抑制MEF2CB及NRP2B的MRNA表現 45
第六節、H2O2造成EDEMA IN PERIDICARDAL SAC的細胞變化 45
第七節、利用MICROARRAY技術分析TAA處理後第24小時對胚胎發育上基因
的影響 47
第八節、結論 47
第五章 討論 49
第一節、肝毒性物TAA對WILD TYPE 斑馬魚胚胎造成EDEMA IN PERICARDIAL SAC 之分析 49
第二節 未來展望 54
第六章、參考文獻 55
第七章、圖表 60
第八章、附錄 83
附錄一、YT&;A保存載體 83
附錄二、PGEM-T表現載體 84

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