紫外光引起DNA損傷其修復過程中間隔填補受酚類化合物延遲的研究

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研究生：	蔡怡芝 Tsai, Yi-Chih
論文名稱：	紫外光引起DNA損傷其修復過程中間隔填補受酚類化合物延遲的研究 Delay of gap filling during repair of UV-induced DNA damage by phenolic group containing compounds
指導教授：	劉銀樟 Liu, Yin-Chang
口試委員:	黃海美 Huang, Haimei 王慧菁 Wang, Hui-Ching 方偉宏 Fang, WH 賴金美 Lai, Jin-Mei 李岳倫 Lee, Yueh-Luen
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	101
中文關鍵詞：	核甘酸修護、鹼機修護、秋水仙胺、蜂膠、延遲、間隙填補
外文關鍵詞：	NER, BER, Colcemid, Propolis, Delay, gap filling
相關次數：	點閱：2 下載：0
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在之前的研究中我們已知秋水仙胺會抑制受紫外光照射細胞生長。在更進一步的研究中我們已知秋水仙胺可抑制核甘酸切除修復的間隙修補步驟。但是秋水仙胺是如何抑制修補的這個機制我們依然不十分清楚。在本研究中我們已證明(1)氧化損傷所造成的核修護間隙延遲，可由一些傳統的氧化試劑誘發,例如過氧化氫,甲基萘醌,甲萘醌,我們推論核甘酸修復及鹼機修護共同發生時所造成競爭現象,造成了間隙延遲。(2)在本篇研究中,我們假設蜂膠中的類黃酮化合物被當成短暫的電子攜帶體將電子傳送到氧分子,隨後產生超氧自由基並形成過氧化氫來造成DNA 的氧化損傷。(3)大量的表現增殖細胞核抗原,可以減少抑制間隙填補現象,這個結果表示增殖細胞核抗原在核甘酸修復和鹼基修復都扮演重要的角色,但是連接酶I與瓣狀內切酶並沒有和增殖細胞核抗原有相同的效應。

In previous study, we have known the colcemid inhibit UV-irradiated cell growth. Further, we also find the colcemid hinder the gap filling of nucleotide excision repair (NER). However, how the colcemid hamper the gap filling, the mechanism is still unclear. In this study, we have demonstrated (I) the oxidative DNA damage delayed the gap filling of NER and inhibition of gap filling was also found with typical BER-inducing agents such as hydrogen peroxide, menadione,and methyl methanesulfonate (MMS). We propose that competitionmay occur between NER and BER, which results in delay of gap filling. (II) We also propose that the flavonoids of propolis serve as temporary carriers of electrons received from transition metal ions that are relayed to oxygen molecules to subsequently generate superoxide and H2O2 which can induce oxidative DNA damage. (III) Over expression PCNA can attune the inhibition of gap, this result shows PCNA plays an important role not only in NER but in base excision repair (BER). However, ligase I and Fen-1 did not produce similar effects.

Index………………………………………………………………………………………………….………….3
Abbreviation    7
Chapter 1    8
Background    8
Chapter 2    13
Material and methods    13
1 Cell cultures.    13
2 Chemicals    13
3 UV irradiation.    13
4 Comet assay.    14
5 Comet assay with incubation of enzymes.    14
6. Small interference RNA (sh RNA) of PCNA, Fen-1 and transfection    15
7 Construction PCNA plasmid and generation the PCNA stable clone    16
8 Western blot    16
9 Nucleoid size analysis    17
10 Cell proliferation by Flow cytometry …………………………………..17
11 Detection of Cellular Oxidative Stress.    18
12 Measurement of H2O2 by the Fox Assay.    18
13 Data Analysis    19
Chapter 3    20
Delay of gap filling during nucleotide excision repair by base excision repair    20
1 Introduction    20
2 Results    20
2.1 Propolis and colcemid inhibit gap filling process of NER    20
2.2 Propolis and Colcemid induce Oxidative DNA damage    21
2.3 Repair of propolis-Induced oxidative DNA damage proceeds regardless of preexposure to UV radiation, whereas repair of CPD sites induced by UV is delayed in the presence of propolis    21
2.4 BER Is required for inhibition of gap filling by propolis    22
3 Discussion    23
3.1 Oxidative damage is the major point to inhibit gap filling of NER    23
3.2 Model    23
4 Figures and Figure Legends    25
Chapter 4    39
Induction of oxidative DNA damage by flavonoids of    39
propolis: its mechanism and implication on antioxidant capacity    39
1 Introduction    39
2.1 Propolis Causes Oxidative DNA Damage Only in the Presence of Fe2+ Ions.    41
2.2 Phenolic compounds of the propolis induce oxidative DNA damage    42
2.3 Pre-treatment propolis renders cells more resistant to the subsequent induction of oxidative DNA damage    43
3.1The model of formation H2O2    44
3.2 cells pre-treated with oxidative reagents become more resistant to oxidative stress    45
3.3 Some proteins related with adaptation mechanism    46
4 Figures and Figure Legends    47
Chapter 5    58
PCNA is involved in delay of gap-filling during repair of UV-induced DNA lesions by a subsequent induction of oxidative DNA damage    58
2 Results    59
2.1 Overexpression of Ligase I does not attenuate colcemid-caused delay of gap-filling    59
2.2 Over-expression of PCNA attenuates colcemid-caused delay of               gap-filling    60
2.3 Knockdown of PCNA exacerbates delay of gap-filling.    60
2.4 Knockdown Fen-1 did not delay the repair of H2O2 induced DNA damage    61
3 Discussion    63
4 Figures and Figure Legends    65
Reference    79
Appendix……………………………………………………………………….83



                                

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