皮肤光老化过程中mtDNA突变及氧化应激改变研究

发布时间：2018-09-03 12:00

【摘要】： 由外源性因素(主要为日光中的紫外线,包括长波紫外线和中波紫外线)导致的不同于自然老化的皮肤衰老过程称为光老化。随着全球臭氧层的破坏和紫外线辐射量的增加,光老化出现泛发趋势,皮肤肿瘤的发病率也逐年上升。流行病学调查显示,皮肤肿瘤的高增长率与光老化的全球性流行存在着密切联系。人皮肤在接受一定累积剂量的紫外线辐射后可依次发生重要事件,包括细胞内活性氧簇(reactive oxygen species,ROS)和氧化应激标志物(8—氧—鸟核苷,异前列烷,硝基酪氨酸等)水平升高、细胞核染色体断裂和线粒体DNA(mitochondrial DNA,mtDNA)突变,从而启动皮肤的损伤、衰老、病变甚至癌变过程,因而光老化最主要的后果就是诱发皮肤恶性肿瘤(如恶性黑色素瘤、基底细胞癌等)。如何切断光老化的发生和发展,进而有效针对日光相关性皮肤恶性肿瘤的初始发生环节加以防治,成为相关领域的研究热点之一。以往对皮肤光老化的研究主要集中于组织病理、超显微结构及生化特性领域,揭示了一些与生长、分化有关的基因(如c-myc,c-fos,EGFR等)在皮肤光老化过程中的作用;但mtDNA突变对皮肤光老化的影响研究并不多见。事实上,mtDNA作为细胞内唯一的核外遗传物质,极易受到氧化应激、代谢改变等外源性因素的影响,发生突变并逐渐累积,最终导致一系列退行性病变和早衰症状的发生。因而近年来,mtDNA突变与皮肤衰老之间的联系已经引起关注。目前认为,光老化过程中可能存在着mtDNA突变累积现象,且其突变是氧化应激和细胞凋亡异常的中间环节。尽管已有的研究在一定程度上揭示了mtDNA突变与皮肤老化之间的联系,但mtDNA突变导致光老化的分子生物学机制,目前尚未阐明。为深入探究mtDNA突变引发皮肤光老化的分子机制,本课题进行了如下研究:首先在不同曝光部位皮肤以实时荧光定量聚合酶链反应(Polymerase chainreaction,PCR)检测两种重要的mtDNA大片段缺失突变(4,977bp缺失,又称普通缺失和3,895bp缺失)水平,探讨其与紫外线照射之间的关系;然后在体外培养的皮肤细胞中以中波紫外线(Ultraviolet B,UVB)反复亚毒剂量辐射诱导出应激性提早衰老(stress-induced premature senescence,SIPS)状态,并在这种UVB诱导的SIPS皮肤细胞中观察两种mtDNA缺失突变的累积;在此基础上,观察异黄酮对SIPS皮肤细胞的保护效应及其作用机制。研究结果对进一步开展光老化临床及基础研究具有指导意义和借鉴作用。 1人皮肤光老化部位的mtDNA大片段缺失突变检测收集不同年龄共71例人皮肤组织,曝光部位(颈项部、手背部或额面部)31例,非曝光部位(臀部、大腿部或腰腹部)40例,分别提取其基因组DNA,对mtDNA中的4,977bp大片段缺失(large deletion of 4,977bp of mtDNA,ΔmtDNA~(4977))和3,895bp大片段缺失(large deletion of 3,895bp of mtDNA,ΔmtDNA~(3895))突变进行扩增,并以荧光实时定量PCR法对两种大片段缺失突变的水平进行检测。结果显示ΔmtDNA~(4977)和ΔmtDNA~(3895)的发生率随年龄而增加,40岁以后人群ΔmtDNA~(4977)和ΔmtDNA~(3895)的发生率显著高于40岁以前人群;ΔmtDNA~(4977)和ΔmtDNA~(3895)的相对拷贝数无论在曝光还是非曝光部位,均与年龄增长呈正相关,且ΔmtDNA~(3895)的累积水平在曝光与非曝光部位相比有显著差异。结果表明ΔmtDNA~(4977)主要与自然老化相关,可作为观察皮肤自然老化进程的指标之一;而ΔmtDNA~(3895)的发生与紫外线照射密切相关,可能是mtDNA针对一定累积剂量的紫外线照射损害所产生的反应,并在皮肤光老化过程中发挥作用。 2 UVB诱导人皮肤细胞进入SIPS状态的模型构建 UVB小剂量、多次照射原代人真皮成纤维细胞(human dermaI fibroblast,HDF)和成人原代表皮角质形成细胞(Human epidermal keratinocyte of adult,HEKa),辐射剂量分别累积达300mJ/cm~2和360mJ/cm~2时,细胞衰老β-半乳糖苷酶(Senescence-associatedβ-galactosidase,SA-β-Gal)化学染色结果呈强阳性,提示被诱导进入衰老状态;流式细胞术(fluorescence-activated cell sorter,FACS)检测到两种皮肤细胞的凋亡率显著上升,大部分细胞被阻滞于G0/G1期;酶联免疫吸附检测(enzyme-liinked immunosorbent assay,ELISA)发现细胞内超氧化物歧化酶(superoxide dismutase,SOD)活性明显下降,丙二醛(malondialdehyde,MDA)含量显著上升。结果表明人皮肤细胞经亚毒剂量UVB重复、多次辐射诱导后进入SIPS状态,可作为一种光损伤、光老化生物学的体外研究模型。 3 SIPS皮肤细胞中mtDNA大片段缺失突变的检测 UVB亚毒剂量、反复照射HDF和HEKa,诱导其进入SIPS状态,提取基因组DNA,以普通PCR检测ΔmtDNA~(4977)和ΔmtDNA~(3895)的发生频率,荧光实时定量PCR检测其突变水平。两种细胞株中ΔmtDNA~(4977)和ΔmtDNA~(3895)的发生频率和表达水平均随UVB照射剂量的增多而逐渐升高;ΔmtDNA~(3895)的发生频率和突变水平最高比ΔmtDNA~(4977)能更灵敏地反映UVB的累积辐射。结果表明mtDNA大片段缺失突变的形成与累积与皮肤细胞接受的累积UV照射剂量密切相关,其中ΔmtDNA~(3895)可作为一种皮肤细胞光损伤的新的生物学观察指标。 4异黄酮对UVB诱导HDF进入SIPS状态的保护作用研究 HDF分别以0,10,20,40和80μmol/L浓度的异黄酮预处理,然后接受累积剂量300mJ/cm~2的UVB辐射,检测各种SIPS相关表型指标以评估异黄酮对SIPS状态的保护效应。结果显示异黄酮可剂量依赖性地抑制SA-β-Gal活化,降低凋亡率并促使细胞周期进入S期,同时降低细胞内MDA水平,增强SOD活性,表明其具有保护HDF进入SIPS状态的作用。异黄酮在较大剂量(80μmol/L)时可降低HDF的ΔmtDNA~(4977)和△mtDNA~(3895)水平,并抑制一种重要的氧化还原蛋白p66Shc(66-kilodalton isoform of the growth factor adapter Shc,生长因子配体Shc的66KD同工蛋白)及其下游信号蛋白FKHRL1(forkhead homolog like 1,叉头样蛋白1活化),提示其可能通过下调线粒体信号通路发挥拮抗氧化应激的效应,从而保护HDF进入SIPS状态。结论 1、ΔmtDNA~(4977)与自然老化相关,是一种皮肤自然老化的指标;而ΔmtDNA~(3895)与紫外线照射密切相关,可视为皮肤光损伤的新的生物学监测指标之一。 2、HDF和HEKa被累积辐射剂量300mJ/cm~2和360mJ/cm~2的UVB小剂量、多次照射诱导进入SIPS状态后,可作为光老化光生物学研究的体外模拟模型之一。 3、异黄酮可剂量依赖性地保护UVB诱导的HDF进入SIPS状态,其作用机制涉及p66Shc-线粒体信号-氧化应激信号通路的下调。
[Abstract]:The process of skin aging caused by exogenous factors (mainly ultraviolet rays in sunlight, including long-wave ultraviolet and medium-wave ultraviolet rays) is called photoaging, which is different from natural aging. With the destruction of the global ozone layer and the increase of ultraviolet radiation, photoaging appears a general trend, and the incidence of skin tumors increases year by year. The high growth rate of skin tumors is closely related to the global epidemic of photoaging. Important events, including reactive oxygen species (ROS) and oxidative stress markers (8-O-guanosine, isoprostane, nitro, etc., can occur in turn after exposure to a certain cumulative dose of ultraviolet radiation. Increased levels of tyrosine, nuclear chromosome breakage, and mitochondrial DNA (mtDNA) mutations trigger skin damage, aging, pathological changes and even canceration. The main consequence of photoaging is to induce skin malignancies (such as malignant melanoma, basal cell carcinoma, etc.). How to cut off the occurrence and development of photoaging It has become one of the research hotspots in related fields to effectively prevent and treat the initial stage of sunlight-related skin malignancies.
Previous studies on skin photoaging have focused on the fields of histopathology, ultrastructure and biochemical characteristics, revealing the role of some genes related to growth and differentiation (e.g. c-myc, c-fos, EGFR, etc.) in the process of skin photoaging; however, there are few studies on the effect of mtDNA mutation on skin photoaging. 1. Extranuclear genetic material is susceptible to oxidative stress, metabolic changes and other exogenous factors. It mutates and accumulates gradually, eventually leading to a series of degenerative diseases and premature aging symptoms. The accumulation of mtDNA mutations is an intermediate link between oxidative stress and abnormal cell apoptosis. Although the relationship between mtDNA mutations and skin aging has been revealed to some extent, the molecular biological mechanism of mtDNA mutations leading to photoaging has not yet been elucidated.
In order to explore the molecular mechanism of skin photoaging induced by mtDNA mutation, the following studies were carried out: First, real-time fluorescence quantitative polymerase chain reaction (PCR) was used to detect two important mtDNA large fragment deletion mutations (4,977 BP deletion, also known as common deletion and 3,895 BP deletion) levels in different exposure sites of skin. Then stress-induced premature senescence (SIPS) was induced by repeated subtoxic dose of Ultraviolet B (UVB) in cultured skin cells, and two mtDNA deletion mutations were observed in SIPS skin cells induced by UVB. On this basis, the protective effect of isoflavones on SIPS skin cells and its mechanism were observed. The results of this study will be helpful for further clinical and basic research on photoaging.
Detection of mtDNA large fragment deletion in 1 skin photoaging sites
Genomic DNA was extracted from 71 human skin tissues of different ages, 31 exposed sites (neck, back of hand or forehead), 40 unexposed sites (buttocks, thighs or waist and abdomen), and 4,977 BP large deletion of mtDNA (large deletion of 4,977 BP of mtDNA, mtDNA ~ (4977)) and 3,895 BP large deletion were detected. The mutations of 3,895 BP of mtDNA and mtDNA ~ (3895) were amplified and the levels of two large deletion mutations were detected by real-time quantitative PCR. The results showed that the incidence of mtDNA ~ (4977) and mtDNA ~ (3895) increased with age. The incidence of mtDNA ~ (4977) and mtDNA ~ (3895) in people aged 40 was significantly higher than those aged 40. The relative copies of mtDNA ~ (4977) and mtDNA ~ (3895) were positively correlated with age at exposed and non-exposed sites, and the cumulative levels of mtDNA ~ (3895) were significantly different between exposed and non-exposed sites. The occurrence of mtDNA ~ (3895) is closely related to ultraviolet radiation, which may be the response of mtDNA to a certain cumulative dose of ultraviolet radiation damage and play a role in the process of skin photoaging.
Model construction of 2 UVB inducing human skin cells to enter SIPS state
Human dermal fibroblast (HDF) and human epidermal keratinocyte of adult (HEKa) were irradiated by UVB at low doses for several times. When the radiation dose accumulated to 300 mJ/cm~2 and 360 mJ/cm~2 respectively, senescence-associated beta-galactosidase (SA-beta-Ga-Ga) was observed. L) The results of chemical staining were strongly positive, suggesting that the cells were induced into senescence; the apoptosis rate of the two kinds of skin cells was significantly increased by flow cytometry (FACS), and most of the cells were blocked in G0/G1 phase; enzyme-linked immunosorbent assay (ELISA) found that the cells were superoxide. Superoxide dismutase (SOD) activity decreased significantly, malondialdehyde (MDA) content increased significantly. The results showed that human skin cells repeat subtoxic dose of UVB and enter SIPS state after repeated radiation. It can be used as an in vitro study model of photodamage and photoaging biology.
Detection of mtDNA large fragment deletion mutation in 3 SIPS skin cells
UVB subtoxic dose, repeated exposure to HDF and HEKa, induced them into SIPS state, extracted genomic DNA, detected the occurrence frequency of mtDNA ~ (4977) and mtDNA ~ (3895) by ordinary PCR, and detected the mutation level by real-time fluorescence quantitative PCR. The occurrence frequency and expression level of mtDNA ~ (4977) and mtDNA ~ (3895) in the two cell lines increased with the dose of UVB irradiation. The occurrence frequency and mutation level of mtDNA ~ (3895) were more sensitive than that of mtDNA ~ (4977) to reflect the cumulative UVB radiation. New biological indicators.
Protective effects of 4 isoflavones on UVB induced HDF entry into SIPS
HDF was pretreated with isoflavones at concentrations of 0,10,20,40 and 80 micromol/L respectively, and then subjected to cumulative dose of 300 mJ/cm~2 UVB radiation. The protective effects of isoflavones on SIPS were evaluated by measuring the phenotypic parameters related to SIPS. Isoflavones at higher doses (80 micromol/L) can reduce the levels of DeltamtDNA ~ (4977) and delta mtDNA ~ (3895) and inhibit an important redox protein p66Shc (66-kilodalton isoform of the growth factor adapter Shc. Growth factor ligand Shc's 66KD isoprotein and its downstream signal protein FKHRL1 (forkhead homolog like 1, forkhead like 1) are activated, suggesting that it may play an antagonistic role in antioxidant stress by down-regulating mitochondrial signaling pathway, thereby protecting HDF from entering SIPS.
conclusion
1. mtDNA ~ (4977) is related to natural aging and is an indicator of skin natural aging, while mtDNA ~ (3895) is closely related to ultraviolet radiation, which can be regarded as one of the new biological monitoring indicators of skin photodamage.
2. HDF and HEKa can be used as one of the in vitro models for photoaging photobiology research after they are induced into SIPS state by repeated irradiation at low doses of UVB with cumulative radiation dose of 300 mJ/cm~2 and 360 mJ/cm~2.
3. Isoflavones can protect HDF from SIPS induced by UVB in a dose-dependent manner by down-regulation of p66Shc-mitochondrial signaling-oxidative stress signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R751

【相似文献】