【摘要】： 由外源性因素(主要為日光中的紫外線,包括長波紫外線和中波紫外線)導致的不同于自然老化的皮膚衰老過程稱為光老化。隨著全球臭氧層的破壞和紫外線輻射量的增加,光老化出現(xiàn)泛發(fā)趨勢,皮膚腫瘤的發(fā)病率也逐年上升。流行病學調(diào)查顯示,皮膚腫瘤的高增長率與光老化的全球性流行存在著密切聯(lián)系。人皮膚在接受一定累積劑量的紫外線輻射后可依次發(fā)生重要事件,包括細胞內(nèi)活性氧簇(reactive oxygen species,ROS)和氧化應激標志物(8—氧—鳥核苷,異前列烷,硝基酪氨酸等)水平升高、細胞核染色體斷裂和線粒體DNA(mitochondrial DNA,mtDNA)突變,從而啟動皮膚的損傷、衰老、病變甚至癌變過程,因而光老化最主要的后果就是誘發(fā)皮膚惡性腫瘤(如惡性黑色素瘤、基底細胞癌等)。如何切斷光老化的發(fā)生和發(fā)展,進而有效針對日光相關性皮膚惡性腫瘤的初始發(fā)生環(huán)節(jié)加以防治,成為相關領域的研究熱點之一。 以往對皮膚光老化的研究主要集中于組織病理、超顯微結構及生化特性領域,揭示了一些與生長、分化有關的基因(如c-myc,c-fos,EGFR等)在皮膚光老化過程中的作用;但mtDNA突變對皮膚光老化的影響研究并不多見。事實上,mtDNA作為細胞內(nèi)唯一的核外遺傳物質,極易受到氧化應激、代謝改變等外源性因素的影響,發(fā)生突變并逐漸累積,最終導致一系列退行性病變和早衰癥狀的發(fā)生。因而近年來,mtDNA突變與皮膚衰老之間的聯(lián)系已經(jīng)引起關注。目前認為,光老化過程中可能存在著mtDNA突變累積現(xiàn)象,且其突變是氧化應激和細胞凋亡異常的中間環(huán)節(jié)。盡管已有的研究在一定程度上揭示了mtDNA突變與皮膚老化之間的聯(lián)系,但mtDNA突變導致光老化的分子生物學機制,目前尚未闡明。 為深入探究mtDNA突變引發(fā)皮膚光老化的分子機制,本課題進行了如下研究:首先在不同曝光部位皮膚以實時熒光定量聚合酶鏈反應(Polymerase chainreaction,PCR)檢測兩種重要的mtDNA大片段缺失突變(4,977bp缺失,又稱普通缺失和3,895bp缺失)水平,探討其與紫外線照射之間的關系;然后在體外培養(yǎng)的皮膚細胞中以中波紫外線(Ultraviolet B,UVB)反復亞毒劑量輻射誘導出應激性提早衰老(stress-induced premature senescence,SIPS)狀態(tài),并在這種UVB誘導的SIPS皮膚細胞中觀察兩種mtDNA缺失突變的累積;在此基礎上,觀察異黃酮對SIPS皮膚細胞的保護效應及其作用機制。研究結果對進一步開展光老化臨床及基礎研究具有指導意義和借鑒作用。 1人皮膚光老化部位的mtDNA大片段缺失突變檢測 收集不同年齡共71例人皮膚組織,曝光部位(頸項部、手背部或額面部)31例,非曝光部位(臀部、大腿部或腰腹部)40例,分別提取其基因組DNA,對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))突變進行擴增,并以熒光實時定量PCR法對兩種大片段缺失突變的水平進行檢測。結果顯示ΔmtDNA~(4977)和ΔmtDNA~(3895)的發(fā)生率隨年齡而增加,40歲以后人群ΔmtDNA~(4977)和ΔmtDNA~(3895)的發(fā)生率顯著高于40歲以前人群;ΔmtDNA~(4977)和ΔmtDNA~(3895)的相對拷貝數(shù)無論在曝光還是非曝光部位,均與年齡增長呈正相關,且ΔmtDNA~(3895)的累積水平在曝光與非曝光部位相比有顯著差異。結果表明ΔmtDNA~(4977)主要與自然老化相關,可作為觀察皮膚自然老化進程的指標之一;而ΔmtDNA~(3895)的發(fā)生與紫外線照射密切相關,可能是mtDNA針對一定累積劑量的紫外線照射損害所產(chǎn)生的反應,并在皮膚光老化過程中發(fā)揮作用。 2 UVB誘導人皮膚細胞進入SIPS狀態(tài)的模型構建 UVB小劑量、多次照射原代人真皮成纖維細胞(human dermaI fibroblast,HDF)和成人原代表皮角質形成細胞(Human epidermal keratinocyte of adult,HEKa),輻射劑量分別累積達300mJ/cm~2和360mJ/cm~2時,細胞衰老β-半乳糖苷酶(Senescence-associatedβ-galactosidase,SA-β-Gal)化學染色結果呈強陽性,提示被誘導進入衰老狀態(tài);流式細胞術(fluorescence-activated cell sorter,FACS)檢測到兩種皮膚細胞的凋亡率顯著上升,大部分細胞被阻滯于G0/G1期;酶聯(lián)免疫吸附檢測(enzyme-liinked immunosorbent assay,ELISA)發(fā)現(xiàn)細胞內(nèi)超氧化物歧化酶(superoxide dismutase,SOD)活性明顯下降,丙二醛(malondialdehyde,MDA)含量顯著上升。結果表明人皮膚細胞經(jīng)亞毒劑量UVB重復、多次輻射誘導后進入SIPS狀態(tài),可作為一種光損傷、光老化生物學的體外研究模型。 3 SIPS皮膚細胞中mtDNA大片段缺失突變的檢測 UVB亞毒劑量、反復照射HDF和HEKa,誘導其進入SIPS狀態(tài),提取基因組DNA,以普通PCR檢測ΔmtDNA~(4977)和ΔmtDNA~(3895)的發(fā)生頻率,熒光實時定量PCR檢測其突變水平。兩種細胞株中ΔmtDNA~(4977)和ΔmtDNA~(3895)的發(fā)生頻率和表達水平均隨UVB照射劑量的增多而逐漸升高;ΔmtDNA~(3895)的發(fā)生頻率和突變水平最高比ΔmtDNA~(4977)能更靈敏地反映UVB的累積輻射。結果表明mtDNA大片段缺失突變的形成與累積與皮膚細胞接受的累積UV照射劑量密切相關,其中ΔmtDNA~(3895)可作為一種皮膚細胞光損傷的新的生物學觀察指標。 4異黃酮對UVB誘導HDF進入SIPS狀態(tài)的保護作用研究 HDF分別以0,10,20,40和80μmol/L濃度的異黃酮預處理,然后接受累積劑量300mJ/cm~2的UVB輻射,檢測各種SIPS相關表型指標以評估異黃酮對SIPS狀態(tài)的保護效應。結果顯示異黃酮可劑量依賴性地抑制SA-β-Gal活化,降低凋亡率并促使細胞周期進入S期,同時降低細胞內(nèi)MDA水平,增強SOD活性,表明其具有保護HDF進入SIPS狀態(tài)的作用。異黃酮在較大劑量(80μmol/L)時可降低HDF的ΔmtDNA~(4977)和△mtDNA~(3895)水平,并抑制一種重要的氧化還原蛋白p66Shc(66-kilodalton isoform of the growth factor adapter Shc,生長因子配體Shc的66KD同工蛋白)及其下游信號蛋白FKHRL1(forkhead homolog like 1,叉頭樣蛋白1活化),提示其可能通過下調(diào)線粒體信號通路發(fā)揮拮抗氧化應激的效應,從而保護HDF進入SIPS狀態(tài)。 結論 1、ΔmtDNA~(4977)與自然老化相關,是一種皮膚自然老化的指標;而ΔmtDNA~(3895)與紫外線照射密切相關,可視為皮膚光損傷的新的生物學監(jiān)測指標之一。 2、HDF和HEKa被累積輻射劑量300mJ/cm~2和360mJ/cm~2的UVB小劑量、多次照射誘導進入SIPS狀態(tài)后,可作為光老化光生物學研究的體外模擬模型之一。 3、異黃酮可劑量依賴性地保護UVB誘導的HDF進入SIPS狀態(tài),其作用機制涉及p66Shc-線粒體信號-氧化應激信號通路的下調(diào)。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R751

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9 劉伊婷;透過標識 看功效[N];中國質量報;2007年

10 吳艷;尖端科技成就美麗[N];中國服飾報;2006年

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