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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48805

Title: 過氧化氫與巴拉刈誘導之氧化壓力對斑馬魚胚胎中卵黃前質1類似蛋白表現與DNA切割修補活性之影響
Effects of paraquat and hydrogen peroxide-induced oxidative stress on the expression of vitellogenin 1-like proteins and excision repair activities in Zebrafish( Danio rerio) embryos.
Authors: Lai, Po-Ling
賴柏伶
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 過氧化氫;氧化壓力;核酸切割修補作用;巴拉刈;卵黃前質1;斑馬魚
hydrogen peroxide;oxidative stress;nucleotide excision repair;paraquat;vitellogenin 1;zebrafish
Date: 2017
Issue Date: 2018-08-22T06:31:28Z
Abstract: 本實驗室先前研究顯示UV損傷的DNA結合活性在斑馬魚胚胎中呈現高表現量。透過親和性吸附實驗及蛋白質質譜分析發現,兩個30及35 kDa UV傷害辨識蛋白其胺基酸序列類似150 kDa斑馬魚卵黃前質1(Vitellogenin 1; zfVg1),顯示低分子量zfVg1類似蛋白為斑馬魚胚胎中UV傷害辨識活性之成分。以轉錄為基礎之DNA修復實驗也發現,單株抗斑馬魚Vg1抗體可抑制斑馬魚萃取液中核酸切割修補(NER)活性,顯示zfVg1類似蛋白會參與紫外線損傷DNA之NER。本實驗室亦發現氧化壓力可調節NER活性,故本論文以巴拉刈(PQ)及過氧化氫(HP)為氧化壓力來源,探討氧化壓力對NER之影響是否與卵黃前質1類似分子有關。DNA修復實驗顯示, PQ及HP 在濃度30至200 μM時,對NER活性反應有不同影響。當胚胎處理HP 200 μM時,NER活性受明顯抑制其強度降低為對照組之19.8%,而在PQ 200 μM時,NER活性為對照組之90.3%,表明NER可能對不同類型的氧化壓力反應有所差異。反之,即時定量 RT-PCR顯示PQ及HP對DNA損傷辨識蛋白UV-DDB2與XPC之基因表現沒有太大影響。表示PQ和HP並非藉抑制此兩種基因表現而影響NER活性。利用抗斑馬魚Vg1抗體進行免疫分析,與控制組相比時,當胚胎暴露於PQ 和HP可使分子量約35到55-kDa之zfVg1類似蛋白含量增加,且在HP 200 μM時35-kDa蛋白其表現量增加最多,表示當胚胎處理HP時,抑制NER活性與Vg1-like蛋白的異常分解可能有相聯性。
A high expression of UV-damaged-DNA binding activity in zebrafish embryos was repeatedly detected in previous studies and two 30 to 35-kDa polypeptides homologous to the 150-kDa zebrafish vitellogenin 1 (zfVg1) have been identified as components of the embryonic UV-binding activity. Because of the inhibition of nucleotide excision repair (NER) capacity in zebrafish extracts by a monoclonal anti-zfVg1 antibody found in recent studies, low-molecular-weight zfVg1-like polypeptides may also participate in NER of UV-damaged DNA. The goal of this study was to examine if oxidative stress induced by paraquat (PQ) and hydrogen peroxide (HP) would disturb NER and the production of zfVg 1-like molecules in zebrafish embryos. A transcription-based in vitro DNA repair assay showed differential effects of PQ and HP at 30 to 200 μM on NER capacity in zebrafish embryos. NER activities monitored by the up-regulation of UV-suppressed marker cDNA transcription were lowered to 19.8% of control in embryos exposed to 200 μM HP, while these activities remained at 90.3% of control in PQ-exposed embryos. In contrast, gene activities encoding NER-associated DNA damage recognition proteins UV-DDB2 and XPC were not affected by PQ or HP at 200 μM according to real-time RT-PCR. Compared to the extracts of control embryos, higher levels of 35 to 55 kDa polypeptides recognized by the anti-Vg1 monoclonal antibody were repeatedly detected in the extracts of PQ and HP-exposed embryos and the highest level of the 35-kDa zfVg1-like factor was present in the extracts of embryos treated with 200 μM HP, suggesting a link between the inhibition of NER in these embryos and an abnormal pattern of zfVg1 cleavage induced by HP.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G001043B021.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48805
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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