紫外线照射对真皮结构和代谢的影响及氮氧化物Tempol保护作用的研究
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摘要
研究背景:
光老化是指长期紫外线(UV)照射而导致的暴光部位皮肤过早老化,其特征性改变为皮肤增厚,粗糙,晦暗及粗深皱纹,真皮层出现光化性弹力纤维变性,光老化的主要病理改变在真皮层。无论从临床表现,组织病理改变还是发生机制,光老化都是完全不同于皮肤自然衰老的一个病程,是可以防治的。考虑到氧自由基在光老化发生发展中的重要作用,寻找稳定的抗氧化剂增强皮肤的抗氧化防御体系,一直是防治光老化的一条重要思路。氮氧化物是一类新的抗氧化剂并已显示出对UV损伤的一些保护作用,因此,引入氮氧化物保护皮肤免遭UV的各种氧化性损伤,包括皮肤光老化将有一定的应用前景。
目的:
1.了解UV照射对真皮结构和代谢的影响,包括对真皮成纤维细胞结构和增殖的影响,氧化损伤作用,对Ⅰ,Ⅲ型胶原纤维及弹力纤维结构和表达的影响以及对基质金属蛋白酶(MMP)-1,MMP-3表达的影响。
2.探讨氮氧化物Tempol对UV引起的真皮结构和各种代谢改变的保护作用,摸索出合适的作用浓度。
方法:
分为体外细胞试验和动物试验两部分。
第一部分以体外培养的人真皮成纤维细胞为研究对象,同时研究长波紫外线(UVA1,340-400nm)和中波紫外线(UVB,280-320nm)照射对细胞各种功能的影响,以MTT法测定细胞增殖率,以流式细胞仪检测细胞周期,以生化方法测定细胞超氧化物歧化酶(SOD)活性和脂质过氧化物丙二醛(MDA)含量,以半定量逆转录。聚合酶链反应及酶连免疫吸附试验测定MMP-1,MMP-3,Ⅰ型和Ⅲ型前胶原蛋白mRNA或蛋白表达水平。细胞接受一定剂量UVA1或UVB照射同时不加或加入不同浓度的Tempol(0.03,0.5,8mM),探讨Tempol对上述各项指标的影响并摸索出合适的作用浓度。
第二部分首先以模拟日光光源(SSR)摸索建立豚鼠皮肤的光老化模型,以苏木素-伊红染色和电镜观察了解豚鼠光老化皮肤真皮结构的变化,以生化方法
Background:Photoaging is due to chronic ultraviolet radiation (UV) which contributes to a premature ageing phenotype of skin at sun-exposed area and characterized by coarsely wrinkled, yellowish skin in clinical appearance and "solar elastosis" change of dermis in histological appearance. It is a complex pathological process with the major damage seen in the connective tissue of the dermis and completely different from intrinsical ageing as related to morphological phenotype and underlying mechanisms, so photoaging can be prevented. Considering the important role of reactive oxygen species in the pathogenesis of photoaging. supplement with exogenic antioxidants to strengthen the antioxidant system of the skin is always one of important strategies to protect against photoaging. Since nitroxide has been reported to have antioxidant and some photoprotective properties, it may be hopeful to import this agent to fight against photoaging.Objective:1. To study the influence of UV on the dermal structure and metabolism, including the structure and proliferation of dermal fibroblasts. the oxidative damage, the structure and expression of collagen I, collagen III. elastic fiber and the expression of matrix metalloproteinase (MMP)-1. MMP-3.2. To determine the protective effects of Tempol, one of nitroxides. against ultraviolet irradiation and to confirm its proper concentration.Methods:The research was dividednto in -vitro study and animal study.Human dermal fibroblasts were used in the in-vitro study. Fibroblasts were irradiated by a single exposure to UVAI (340-400nm) or UVB (280-320nm) and at the same time incubated with, or without. Tempol and detected twenty-four hours later. Cell
proliferation rate was determined by MTT assay and cell cycling was analyzed by flow cytometric method. Superoxide dismutase (SOD) activity and lipid peroxidation, as shown by accumulation malonyldialdehyde (MDA). were detected by biochemical assay. Expressions of procollagen I. procollagen 111 (protein levels) and MMP-1, MMP-3 (mRNA level) were detected by ELISA and semi-quantitative reverse transcriptase-PCR separately.In animal study, the guinea pig photoaging model with chronic sun-simulating radiation (SSR) exposure was set up firstly. The protective effects of Tempol, topically used before each exposure at the concentration of 5 mg/ml or 0.5 mg/ml, were also assessed. Dermal structure was observed after Hematoxylin-eosin stain or by electron microscopy. SOD activity and MDA level in the skin were detected by biochemical assay. The structure and the expression of elastic fiber were analyzed after Weigert's stain and the structure of collagen 1 and collagen III was observed after Siriured's stain by polarized microscopy.Results:In in-vitro study, cell proliferation curve after UVA1 or UVB irradiation showed dose dependent decrement pattern. More cells stopped at the GO/GI phase and the proliferation index (PI) decreased after 15 J/cm2UVAl irradiation. 15 J/cm2UVAl or 40 mJ/cm2 UVB significantly inhibited SOD activity and procollagen I, procollagen III protein levels, while increased MDA level and stimulated MMP-1 and MMP-3 mRNA expression. Tempol, between 0.03 mM and 8 mM, reversed these effects caused by UVA1 or UVB in some degree or even completely and in proper concentration, the results were statistically significant compared with irradiated group. Tempol at concentration of 0.5 mM had strongest photoprotective effects for UVA1 or UVB exposed human dermal fibroblasts and was even more effective than traditional antioxidant vitamin C (0.1%).In animal study, after depilated by rosin and beeswax mixture, albino guinea pigs were subjected to suberythemal dose of SSR three times a week. Seventeen weeks after exposure, there was typical "solar elastosis" damage in the upper dermis. Dermal fibroblasts appeared metabolically hyperactive and mitochondria in the cells were damaged. Some cells even broke up. Mature elastic fibers and collagen were severely degraded and there was large amount of elastotic material accumulated in the subepidermal dermis. SSR irradiation also provoked an infiltration into the dermis of
光老化是指长期紫外线(UV)照射而导致的暴光部位皮肤过早老化,其特征性改变为皮肤增厚,粗糙,晦暗及粗深皱纹,真皮层出现光化性弹力纤维变性,光老化的主要病理改变在真皮层。无论从临床表现,组织病理改变还是发生机制,光老化都是完全不同于皮肤自然衰老的一个病程,是可以防治的。考虑到氧自由基在光老化发生发展中的重要作用,寻找稳定的抗氧化剂增强皮肤的抗氧化防御体系,一直是防治光老化的一条重要思路。氮氧化物是一类新的抗氧化剂并已显示出对UV损伤的一些保护作用,因此,引入氮氧化物保护皮肤免遭UV的各种氧化性损伤,包括皮肤光老化将有一定的应用前景。
目的:
1.了解UV照射对真皮结构和代谢的影响,包括对真皮成纤维细胞结构和增殖的影响,氧化损伤作用,对Ⅰ,Ⅲ型胶原纤维及弹力纤维结构和表达的影响以及对基质金属蛋白酶(MMP)-1,MMP-3表达的影响。
2.探讨氮氧化物Tempol对UV引起的真皮结构和各种代谢改变的保护作用,摸索出合适的作用浓度。
方法:
分为体外细胞试验和动物试验两部分。
第一部分以体外培养的人真皮成纤维细胞为研究对象,同时研究长波紫外线(UVA1,340-400nm)和中波紫外线(UVB,280-320nm)照射对细胞各种功能的影响,以MTT法测定细胞增殖率,以流式细胞仪检测细胞周期,以生化方法测定细胞超氧化物歧化酶(SOD)活性和脂质过氧化物丙二醛(MDA)含量,以半定量逆转录。聚合酶链反应及酶连免疫吸附试验测定MMP-1,MMP-3,Ⅰ型和Ⅲ型前胶原蛋白mRNA或蛋白表达水平。细胞接受一定剂量UVA1或UVB照射同时不加或加入不同浓度的Tempol(0.03,0.5,8mM),探讨Tempol对上述各项指标的影响并摸索出合适的作用浓度。
第二部分首先以模拟日光光源(SSR)摸索建立豚鼠皮肤的光老化模型,以苏木素-伊红染色和电镜观察了解豚鼠光老化皮肤真皮结构的变化,以生化方法
Background:Photoaging is due to chronic ultraviolet radiation (UV) which contributes to a premature ageing phenotype of skin at sun-exposed area and characterized by coarsely wrinkled, yellowish skin in clinical appearance and "solar elastosis" change of dermis in histological appearance. It is a complex pathological process with the major damage seen in the connective tissue of the dermis and completely different from intrinsical ageing as related to morphological phenotype and underlying mechanisms, so photoaging can be prevented. Considering the important role of reactive oxygen species in the pathogenesis of photoaging. supplement with exogenic antioxidants to strengthen the antioxidant system of the skin is always one of important strategies to protect against photoaging. Since nitroxide has been reported to have antioxidant and some photoprotective properties, it may be hopeful to import this agent to fight against photoaging.Objective:1. To study the influence of UV on the dermal structure and metabolism, including the structure and proliferation of dermal fibroblasts. the oxidative damage, the structure and expression of collagen I, collagen III. elastic fiber and the expression of matrix metalloproteinase (MMP)-1. MMP-3.2. To determine the protective effects of Tempol, one of nitroxides. against ultraviolet irradiation and to confirm its proper concentration.Methods:The research was dividednto in -vitro study and animal study.Human dermal fibroblasts were used in the in-vitro study. Fibroblasts were irradiated by a single exposure to UVAI (340-400nm) or UVB (280-320nm) and at the same time incubated with, or without. Tempol and detected twenty-four hours later. Cell
proliferation rate was determined by MTT assay and cell cycling was analyzed by flow cytometric method. Superoxide dismutase (SOD) activity and lipid peroxidation, as shown by accumulation malonyldialdehyde (MDA). were detected by biochemical assay. Expressions of procollagen I. procollagen 111 (protein levels) and MMP-1, MMP-3 (mRNA level) were detected by ELISA and semi-quantitative reverse transcriptase-PCR separately.In animal study, the guinea pig photoaging model with chronic sun-simulating radiation (SSR) exposure was set up firstly. The protective effects of Tempol, topically used before each exposure at the concentration of 5 mg/ml or 0.5 mg/ml, were also assessed. Dermal structure was observed after Hematoxylin-eosin stain or by electron microscopy. SOD activity and MDA level in the skin were detected by biochemical assay. The structure and the expression of elastic fiber were analyzed after Weigert's stain and the structure of collagen 1 and collagen III was observed after Siriured's stain by polarized microscopy.Results:In in-vitro study, cell proliferation curve after UVA1 or UVB irradiation showed dose dependent decrement pattern. More cells stopped at the GO/GI phase and the proliferation index (PI) decreased after 15 J/cm2UVAl irradiation. 15 J/cm2UVAl or 40 mJ/cm2 UVB significantly inhibited SOD activity and procollagen I, procollagen III protein levels, while increased MDA level and stimulated MMP-1 and MMP-3 mRNA expression. Tempol, between 0.03 mM and 8 mM, reversed these effects caused by UVA1 or UVB in some degree or even completely and in proper concentration, the results were statistically significant compared with irradiated group. Tempol at concentration of 0.5 mM had strongest photoprotective effects for UVA1 or UVB exposed human dermal fibroblasts and was even more effective than traditional antioxidant vitamin C (0.1%).In animal study, after depilated by rosin and beeswax mixture, albino guinea pigs were subjected to suberythemal dose of SSR three times a week. Seventeen weeks after exposure, there was typical "solar elastosis" damage in the upper dermis. Dermal fibroblasts appeared metabolically hyperactive and mitochondria in the cells were damaged. Some cells even broke up. Mature elastic fibers and collagen were severely degraded and there was large amount of elastotic material accumulated in the subepidermal dermis. SSR irradiation also provoked an infiltration into the dermis of
引文
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[2] Fisher GJ, Datta SC, Talwar HS, et al. Molecular basis of sun-induced premature skin ageing and retinoid antagonism[J] . Nature, 1996, 379(6563): 335-339.
[3] Fisher GJ, Talwar HS, Lin J, et al. Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo[J]. J Clin Invest, 1998, 101(6): 1432-1440.
[4] Brenneisen P, Wenk J, KIotz LO, et al. Central role of Ferrous/Ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial collagenase (matrix-degrading metalloprotease (MMP)-1) and stromelysin-1 (MMP-3) mRNA levels in cultured human dermal fibroblasts[J]. J Biol Chem, 1998, 273(9): 5279-5287.
[5] Kang S, Chung JH, Lee JH, et al. Topical N-acetyl cysteine and genistein prevent ultraviolet-light-induced signaling that leads to photoaging in human skin in vivo[J]. J Invest Dermatol, 2003, 120(5): 835-841.
[6] Ma W, Wlaschek M, Tantcheva-Poor 1, et al. Chronological ageing and photoageing of the fibroblasts and the dermal connective tissue[J]. Clin Exp Dermatol, 2001, 26(7): 592-599.
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