臺灣博碩士論文加值系統

已知皮膚長期受到UVA的照射會有致癌的危險。有研究指出UVA對細胞的傷害主要是活性氧（1O2、O2.-、H2O2或.OH）所致，但UVA照射皮膚細胞可能會誘發NO的生成，NO可能和O2.-反應生成活性較大的peroxynitrite (ONOO.)，而導致細胞受到更嚴重的傷害。依據此點，本研究分為兩部分，分別探討UVA及NO對人類上皮成纖母細胞的傷害以及數種食品、化妝品及細胞成分之作用。
第一部份 近紫外線照射與鐵離子對人類上皮成纖母細胞之氧化傷害
本研究的目的主要是探討Fe2+與氮基三醋酸（NTA）對UVA所誘發的細胞毒性之影響。由實驗結果得知Fe2+與Fe2+-NTA明顯的會促進UVA所導致之膜脂質過氧化(TBARS之生成)、DNA氧化傷害(DNA strand breakage、8-OHdG之生成)與細胞毒性（降低細胞存活率）（p＜0.05）。細胞膜氧化傷害之原因可能為UVA照射細胞所產生的單重態氧（1O2）和細胞膜LH直接反應形成LOOH，Fe2+會依據反應式(1)和LOOH作用
LOOH + Fe2+ → LO. + .OH + Fe3+ (1)
Fe3+被UVA還原成Fe2+，進而持續催化自由基反應，造成脂質嚴重的氧化。DNA傷害之原因可能為Fe2+和UVA照射細胞所生成的H2O2作用產生反應性最強的.OH，因而促進DNA股斷裂及8-OH-dG之生成，最後導致necrosis。而NTA在此試驗模式下明顯的促進Fe2+對Hs68 cells之DNA氧化傷害，可能的原因為NTA改變鐵離子在細胞內存在的形式、分佈或redox potential之改變有關。由實驗的結果發現脂質過氧化與DNA股斷裂之間沒有因果關係，故脂質過氧化與DNA氧化傷害之因果論有待重新評估。
第二部份 數種食品、化妝品及細胞成分之抗氧化能力研究
本研究的目的為探討數種食品（maltol）、化妝品（benzophenone、BM-DBM與boldine）及細胞成分（DMG與2,3-DPG）對UVA及NO所造成的氧化傷害之作用。在UVA傷害方面：這六種皆不具抑制脂質過氧化之能力，其中maltol與BM-DBM卻有促進的作用。但DMG、2,3-DPG與boldine能降低UVA對DNA的傷害，而maltol與BM-DBM卻會促進DNA傷害。在NO的傷害方面：因為NO不會引發脂質過氧化反應，故本部分只探討對DNA傷害之作用。2,3-DPG、benzophenone、BM-DBM及boldine皆能抑制NO所引發之DNA strand breakage，其中以BM-DBM抑制作用最佳。由此推論2,3-DPG與boldine可能可以抵抗UVA與NO所造成的氧化壓力。
結論
綜合以上所述，可知UVA照射會造成Hs68 cells氧化傷害，而Fe2+-NTA會顯著的促進UVA所引發之脂質過氧化與DNA氧化傷害（p＜0.05），其作用機制可能是NTA改變了鐵離子在細胞內存在的形式、分佈或redox potential之改變有關，此種傷害會導致細胞壞死（necrosis）或增加細胞遺傳訊息突變之危害。
已知受到UVA照射之細胞會因DNA受損而突變或癌化，而UVA照射會誘發NO的生成， NO會刺激黑色素細胞產生melanin而造成黑色素沈澱，並會引發發炎反應而導致紅斑的生成。而2,3-DPG與boldine皆能抑制UVA與NO所造成的DNA傷害，故2,3-DPG與boldine可能具有應用為防曬品之潛力。

It is well known that cumulative exposure of human skin to UVA results in increased risk of skin cancer. Some studies indicated that cellular lesion induced by UVA was due to reactive oxygen species (ROS; 1O2, O2.-, H2O2 or .OH). In UVA treated cells, the simultaneous production of NO and ROS might result in the formation of ONOO.- and increasing cellular damage. Therefore, this thesis work was divided into two parts. One was to investigate oxidative damage to human foreskin fibroblasts (Hs68 cells) by UVA and iron ions, the other was to study protective effect of food, cosmetic and cellular components against UVA- or NO-induced oxidative damage in Hs68 cells.
PartⅠ Oxidative damage to Hs68 cells induced by UVA and iron ions
This part of study was to investigate effect of ferrous ion (Fe2+) and nitrilotriacetate (NTA) on cytotoxicity induced by UVA. The results show that Fe2+ and Fe2+-NTA significantly promoted membrane lipid peroxidation (the generation of TBARS was increased), DNA oxidative damage (DNA strand breakage and the generation of 8-hydroxyguanosine (8-OH-dG)) and cytotoxicity (reduction of cellular viability)(p＜0.05). Lipid peroxidation may be caused by singlet oxygen (1O2 ) generated from cells during UVA irradiation, which reacted with membrane lipid (LH) to form lipid hydroxyperoxide (LOOH). The role of iron ions in lipid peroxidation is likely in the breakdown of LOOH according to reaction (1):
LOOH + Fe2+→LO. + .OH + Fe3+ (1)
Fe3+ is thought to be reduced by UVA to Fe2+, which induces the generation of free radical continuously to increase lipid peroxidation. DNA oxidative damage may be caused by reaction of Fe2+ with H2O2 generated from UVA irradiated cells to produce .OH, which induces DNA strand breakage and the formation of 8-OH-dG, leading to necrosis. In the experimental model, NTA significantly promoted DNA oxidative damage in Hs68 cells induced by Fe2+, possibly by chelation of iron ions, leading to changes of distribution and redox potential of cellular iron ions. The result also shows that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+-NTA/cell system.
Part Ⅱ Protective effect of food, cosmetic and cellular components against UVA- or NO-induced oxidative damage in Hs68 cells
This part of study was to investigate the effects of several components of food (maltol), cosmetics (benzophenone, butyl methoxydibenzoylmethane (BM-DBM) and boldine) and mammalian cells (dimethylglycine (DMG) and diphosphoglycerate (DPG)) on oxidative damage induced by UVA or NO. In cellular damage induced by UVA, none of the six compounds inhibited lipid peroxidation. In fact, matol and BM-DBM enhanced lipid peroxidation. DMG, 2,3-DPG and boldine inhibited DNA oxidative damage induced by UVA, but maltol and BM-DBM could enhance it. Cellular damage induced by NO: in the present study, we only investigated DNA oxidative damage induced by NO because NO might not intiate lipid peroxidation. The result shows that 2,3-DPG, benzophenone, BM-DBM and boldine could inhibit DNA strand breakage induced by NO. Among them, BM-DBM had better inhibitory effect. According to it, we suggest that 2,3-DPG and boldine may protect against oxidative stress induced by UVA and NO.
Conclusion
The present results show that Fe2+-NTA strongly enhanced lipid peroxidation and DNA oxidative damage induced by UVA; the mechanism may be that NTA changed the distribution and redox potential of cellular iron ions. Such damage leads to cell death - necrosis, and can increase the risk of mutation of DNA and carcinogenesis.
UVA radiation may induce the generation of NO, which may stimulate melanocytes to generate and accumulate melanin. NO may induce inflammatory response and result in the formation of erythema. 2,3-DPG and boldine can inhibit DNA oxidative damage induced by UVA and NO, and they may have potentials to be sunscreens.

1.前言………………………………………………………………1
1.1緒論…………………………………………………………………...1
1.2第一部份：近紫外線照射與鐵離子對人類上皮成纖母細胞之氧化傷害……………………………………………………………….……..1
1.3第二部份：數種細胞、食品及化妝品成分之抗氧化能力研究……3
1.4 NO 之生理毒性…………………………………………………….…6
1.5研究目標……………………………………………………………... .6
2.材料與方法…………………………………………………….…7
2.1材料…..…………………………………….…………………………….7
2.2抗氧化試驗……………………………………….……………….……...7
2.2.1去氧核醣降解試驗……………………………………………...7
2.2.2螯合亞鐵離子能力之測定……………………………………...8
2.2.3抗脂質過氧化能力之測定……………………………………...9
2.2.4紅血球之製備…………………………………………………...9
2.2.5 TBARS含量之測定…………………………………..……….10
2.2.6溶血程度之測定……………………………………....……….10
2.3 UVA照射劑量之測定……………………..……….………….………..10
2.4細胞培養…………….…………………………..….………….………..10
2.5細胞解凍與保存…….…………………………..….………….………..11
2.6細胞之處理……….…………………………..….…………….………..11
2.6.1 UVA照射處理…….…………… …..….………….………….11
2.6.2 SNP處理…………..…………… …..….………….………….12
2.7細胞存活率分析(MTT viability assay)……..….………….……………12
2.8蛋白質含量之測定…………………...……..….………….……………13
2.9脂質過氧化之測定…………………...……..….………….……………13
2.10 DNA損傷(DNA damage)之測定…...……..….………….……………13
2.10.1 DNA之純化……..…………………………………..……….13
2.10.2 Comet assay (or single cell gel electrophoresis assay)……….13
2.10.3 8-OH-dG含量之測定………………………………..……….14
2.10.4 DNA fragmentation之測定…………………………….…….15
2.11 Histone-associated DNA fragmentation test(HADF)…………………15
2.12統計分析……………………….…...……..….………….……………15
3.結果…….………………………………………………………..16
3.1 UVA與鐵離子對Hs68 cells 之氧化傷害...….………….……………16
3.1.1細胞形態之改變…..…………………………………..……….16
3.1.2細胞之存活率……..…………………………………..……….16
3.1.3脂質過氧化………..…………………………………..……….16
3.1.4 DNA氧化傷害..…..…………………………………..……….16
3.1.4.1 DNA strand breakage…………………………………….16
3.1.4.2 8-OH-dG………………………………………………….17
3.1.4.3 DNA fragmentation…………………………………...….17
3.1.4.4 Histone-associated DNA fragmentation…………………17
3.1.5 BHT對UVA照射與Fe2+-NTA所引發的氧化傷害之影響….18
3.2數種細胞、食品及化妝品成分之抗氧化能力研究..….………….……18
3.2.1 DMG及2,3-DPG螯合鐵離子能力之測定…………..……….18
3.2.2 DMG及2,3-DPG對去氧核醣氧化傷害之影響.……………..18
3.2.3 DMG及2,3-DPG之抗脂質過氧化活性……………..……….19
3.2.4對紅血球脂質過氧化之影響……………..…………..……….19
3.2.5對紅血球溶血之影響……………………..…………..……….20
3.2.6 DMG、2,3-DPG與maltol對UVA/ Fe2+-NTA所引發之氧化傷害的影響…………..…………..…………………………….20
3.2.7 Benzophenone、BM-DBM與Boldine對UVA所引發之氧化傷害的影響…………..…………..…………………………….21
3.2.8 數種細胞、食品及化妝品成分對NO所引發之氧化傷害的影響……………..…………..………………………………….22
4.討論…….………………………………………………………..23
4.1第一部份：UVA照射與鐵離子對Hs68 cells 之氧化傷害 …23
4.1.1脂質過氧化……………………………..……………..……….23
4.1.2 DNA氧化傷害..…..…………………………………..……….23
4.1.3脂質過氧化與 DNA氧化傷害..……………………..……….24
4.1.4 DNA傷害、細胞死亡與基因毒性…………………..……….25
4.2數種細胞、食品及化妝品成分之抗氧化能力研究..….………26
4.2.1 DMG之抗氧化力……………………………………..……….26
4.2.2 2,3-DPG之抗氧化力……………………………………..…26
4.2.3 Maltol之抗氧化力………..…………………………..……….27
4.2.4 Benzophenone、BM-DBM與boldine之抗氧化力……….…27
5.結論.…….……………………………………………………….29
參考文獻….……………………………………………………….31
圖表……….……………………………………………………….44

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