English  |  正體中文  |  简体中文  |  Items with full text/Total items : 26988/38789
Visitors : 2351404      Online Users : 31
RC Version 4.0 © Powered By DSPACE, MIT. Enhanced by NTU Library IR team.
Scope Adv. Search
LoginUploadHelpAboutAdminister

Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48799

Title: 探討過氧化氫及巴拉刈對斑馬魚胚胎中紫外線傷害DNA辨識效應之影響
Differential effects of hydrogen peroxide and paraquat on CPD and 6-4PP binding activities in zebrafish embryos
Authors: Zhuang, Jie
莊婕
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 氧化壓力;過氧化氫;巴拉刈;環丁烷嘧啶雙體;6-4光產物
oxidative stress;hydrogen peroxide;paraquat;CPD;6-4PP
Date: 2017
Issue Date: 2018-08-22T06:31:25Z
Abstract: 核酸剪切修復(Nucleotide Excision Repair, NER)是一種DNA修復系統,用來移除嚴重扭曲之DNA雙螺旋鏈,如DNA受到紫外線(Ultraviolet, UV)照射後,所產生的環丁烷嘧啶雙體(CPDs)、6-4嘧啶光產物(6-4PPs)透過實驗室先前實驗結果發現,將斑馬魚胚胎經過氯化鎘(CdCl2)及巴拉刈(PQ) 的處理會誘導胚胎細胞內活性氧化物上升,使胚胎對CPDs的辨識活性有抑制作用,但對6-4PPs的辨識活性則無明顯影響;以及ROS也會影響NER中辨識蛋白基因的表現,但其作用機制尚未清楚。 在本實驗另外加入過氧化氫(HP)與Cd、PQ來比較,不同的氧化壓力下斑馬魚胚胎蛋白對6-4PP及CPD的辨識活性是否不同。經過9小時30~200 μM PQ處理後,不影響10 hpf 斑馬魚胚胎蛋白粗萃取液(CE)對6-4PP的辨識活性,只有在5 μM Cd與200 μM HP處理下對6-4PP辨識活性有些微抑制;而斑馬魚胚胎蛋白對CPD的辨識活性在Cd、HP與PQ處理下則都會被抑制。實驗再利用即時定量聚合酶連鎖反應(QPCR)來探討,NER系統之辨識蛋白DDB2、XPC的基因表現量與斑馬魚胚胎蛋白對CPD及6-4PP的辨識活性是否相關。結果顯示在100 μM HP、30~100 μM PQ處理下,DDB2基因表現量有些微下降,可能與EMSA結果中被抑制的CPD辨識活性有關;但在5 μM Cd處理下,6-4PP的辨識活性受抑制,可能與XPC的基因表現沒有明顯關聯。 實驗室先前已證實斑馬魚在胚胎初期有Vg1-like蛋白產生,且會對UV損傷DNA具有辨識能力。為了確認Vg1-like蛋白是否參與NER系統中辨識步驟,本實驗利用cross-linking/western blot來分析Vg1-like蛋白是否會直接與UV損傷之DNA進行接觸。當斑馬魚胚胎蛋白與有無照射UV之DNA探針混合並cross-link後,在以anti-Vg1抗體偵測,結果發現小分子量Vg1-like蛋白分子量沒有明顯差別,但70 kDa的Vg1蛋白對有照射UV之DNA探針cross-linking後分子量提升,推測大分子量的Vg1-like蛋白可能會直接與UV損傷DNA接觸。
Nucleotide excision repair (NER) is the main pathway to remove the UV-induced bulky DNA lesions like cyclobutane pyrimidine dimer (CPD) and 6-4 photoproduct (6-4PP). Our earlier studies showed the ability of ROS-inducing agents cadmium (Cd) and paraquat (PQ) to down-regulate CPD binding activities in zebrafish embryos as shown by band shift assay. Exposure of embryos to Cd and PQ, however, caused a weak stimulation of 6-4PP binding activity. The objective of this study was to further explore the differential effects of oxidative stressors on CPD and 6-4PP sensing activities and the possible mechanisms. Exposure of zebrafish embryos at 1 h post fertilization (hpf) to PQ at 30 to 200 μM for 9 hr did not affect 6-4PP binding activity, while hydrogen peroxide (HP) at 200 μM and Cd at 5 μM caused a weak inhibition of this binding activity. The formation of CPD band shifts was inhibited by HP, Cd and PQ at all levels. Hence, HP, Cd and PQ imposed differential inhibitory effects on UV-damaged-DNA binding activities. UV-damaged DNA-binding protein (UV-DDB) and xeroderma pigmentosum C (XPC) are damage recognition factors of NER. Down-regulated gene expression of DDB2 determined by real-time RT-PCR was partially consistent with inhibition of CPD binding activity when zebrafish embryos were exposed to HP at 100 μM and PQ at 30 to 100 μM. No close association between inhibition of 6-4PP binding activity and XPC gene expression was observed in embryos exposed to 5 μM Cd. Participation of low-molecular-weight homologs of the 150-kDa zebrafish vitellogenin1 (Vg1) in NER has been identified in our previous studies. To explore the possible involvement of Vg1-ike protein in the damage recognition stage of NER, UV crosslinking was conducted to examine if Vg1-like factors contacted UV-damaged DNA. Crosslinking assay showed no changes in the molecular weights of Vg1-like proteins based on immunoblot analysis of Vg1-like proteins present in zebrafish extracts containing UV-irradiated or unirradiated DNA probe. No crosslinking of low-molecular-weight Vg1-like factors to CPDs or 6-4PPs was observed under oxidative stress. How Vg1-like factors participate in NER needs further study.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G001053B001.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48799
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

Files in This Item:

File Description SizeFormat
index.html0KbHTML15View/Open


All items in NTOUR are protected by copyright, with all rights reserved.

 


著作權政策宣告: 本網站之內容為國立臺灣海洋大學所收錄之機構典藏,無償提供學術研究與公眾教育等公益性使用,請合理使用本網站之內容,以尊重著作權人之權益。
網站維護: 海大圖資處 圖書系統組
DSpace Software Copyright © 2002-2004  MIT &  Hewlett-Packard  /   Enhanced by   NTU Library IR team Copyright ©   - Feedback