【作者】 王继华；

【导师】 鲁开化； 赵亚南；

【作者基本信息】 昆明医学院 ， 外科学， 2008， 博士

【摘要】 研究背景及目的皮肤老化分内源性老化和外源性老化,内源性因素引起的皮肤老化是不可避免的自然过程,其具体机制尚不清楚。在外源性因素中,紫外线的作用是最主要和最突出的因素,由于皮肤反复暴露于紫外线中造成的皮肤老化称为光老化(Photoaging)。光老化的皮肤发生结构和功能的特征性改变,临床上这些特征性改变包括:皱纹、皮肤松弛、粗糙萎缩、斑点状色素沉着、雀斑、毛细血管扩张、甚至癌变。成纤维细胞是真皮中最主要的细胞成分,紫外线(UV)照射导致成纤维细胞的过早衰老,胶原合成能力下降,基质金属蛋白酶(MMPs)表达升高,细胞外基质降解是皮肤光老化的最重要原因。紫外线依其波长分为短波紫外线UVC(200～280 nm)、中波紫外线UVB(280～320 nm)和长波紫外线UVA(320～400 nm)。99%的UVA到达地球表面,超过50%的UVA穿透表皮到达真皮层,作用于成纤维细胞。因此,UVA与人体皮肤关系密切,在皮肤光老化的发生日显重要,并受到各国公共卫生学者的关注。在皮肤光老化发生的机制方面,因UV照射产生氧自由基学说是目前国内外学者普遍承认的衰老理论之一,因而,近年来国内外学者针对UVA照射产生的氧自由基进行了大量的体内外抗氧化研究,但至今尚未找到确切的抗光老化方法。随着对光老化发生机制的研究深入,研究者发现细胞因子之间的调控对皮肤光老化起着很重要的作用,细胞因子在光老化中的作用越来越受到重视。但从细胞因子角度抗皮肤光老化的研究报道甚少。本研究旨在探讨不同剂量UVA照射体外培养的皮肽成纤维细胞Ⅰ型、Ⅲ型胶原合成和TGF-β1表达情况;探讨加入TGF-β1对胶原合成的影响;TGF-β1对UVA照射体外培养的皮肤成纤维细胞MMP-1、MMP-3高表达有无抑制作用;对UVA照射导致的线粒体脱氧核糖核酸(mtDNA)缺失有无保护作用,以及TGF-β1对热休克蛋白70(HSP70)表达的影响,全面系统的探讨TGF-β1对UVA照射体外培养皮肤成纤维细胞光老化的保护作用,以及TGF-β1对UVA照射体外培养的皮肤成纤维细胞IGF-1、KGF,VEGF分泌的影响。研究方法:1.取年轻成人包皮成纤维细胞培养传代,用第5代细胞做实验对象,进行细胞分组。采用不同计量的UVA(5 J/cm~2、10J/cm~2、20J/cm~2)照射,设立对照组(0 J/cm~2.),建立成纤维细胞光老化模型。培养24h后采用四甲基偶氮唑盐比色(MTT)法检测照射后细胞增殖活性,酶联免疫(ELISA)法测定UVA照射皮肤成纤维细胞上清液中Ⅰ型、Ⅲ型胶原和TGF-β1蛋白含量,半定量RT—PCR检测Ⅰ型、Ⅲ型胶原mRNA、TGF-β1mRNA表达水平。2.在培养液中分别加入不同剂量TGF-β1,即小剂量(0.1ng/ml)、中剂量(1.0 ng/ml)、大剂量(10.0 ng/ml),用UVA15J/cm~2照射,采用酶联免疫(ELISA)法测定TGF-β1对UVA照射皮肤成纤维细胞上清液中Ⅰ型、Ⅲ型胶原含量,HSP70表达变化;半定量RT—PCR检测Ⅰ型、Ⅲ型胶原mRNA和MMP-1、MMP-3 mRNA、Smad3 mRNA表达变化,线粒体DNA(mtDNA)4977bp缺失情况。采用酶联免疫(ELISA)法测定TGF-β1对UVA照射体外培养的皮肤成纤维细胞上清液中IGF-1、KGF、VEGF含量的影响。研究结果:1.不同剂量的UVA(5 J/cm~2、10J/cm~2、20J/cm~2)照射体外培养的皮肤成纤维细胞结果显示:随着UVA照射剂量增加,成纤维细胞OD值下降,与对照组比较,UVA 10 J/cm~2、UVA 20 J/cm~2照射组OD值明显下降,有非常显著性差异(P＜0.01)。Ⅰ型胶原含量和mRNA表达,随着UVA照射剂量增加而降低,呈剂量依赖关系。与对照组比较,UVA 10 J/cm~2、UVA 20 J/cm~2照射组降低明显(P＜0.05,P＜0.01),UVA 20 J/cm~2组抑制胶原合成作用更加明显,与对照组及其它两组比较,有非常显著性差异(P＜0.01)。Ⅲ型胶原含量和mRNA表达,与对照组比较,UVA 20 J/cm~2照射组明显降低,有非常显著性差异(P＜0.01)。TGF-β1蛋白含量和mRNA表达随着UVA照射剂量增加而降低,呈剂量依赖关系。与对照组比较,UVA 10 J/cm~2、UVA 20 J/cm~2照射组,TGF-β1蛋白含量和mRNA表达明显降低,有非常显著性差异(P＜0.01)。2.本实验通过UVA15 J/cm~2照射不同剂量TGF-β1处理后的成纤维细胞,结果显示:随着TGF-β1剂量增加,大、中剂量组成纤维细胞OD值明显高于UVA照射组,有非常显著性差异(P＜0.01)。Ⅰ型、Ⅲ型胶原蛋白含量随着TGF-β1剂量增加而增加,呈剂量依赖关系。大、中剂量组与UVA照射组比较,具有显著性差异(P＜0.01,P＜0.05),大剂量组促进胶原合成作用更明显,有非常显著性差异(P＜0.01)。Ⅰ型、Ⅲ型胶原mRNA表达,随着TGF-β1剂量增加而升高,呈剂量依赖关系。Ⅰ型胶原mRNA表达在大、中剂量组明显升高,与UVA照射组比较,有非常显著性差异(P＜0.01)。Ⅲ型胶原mRNA表达,大剂量组与UVA照射组比较,有非常显著性差异(P＜0.01)。不同剂量UVA照射后MMP-1、MMP-3mRNA表达与对照组比较,UVA 10J/cm~2、UVA 20 J/cm~2照射组,MMP-1、MMP-3mRNA表达明显升高,有显著性差异(P＜0.05,P＜0.01),UVA 20 J/cm~2组MMP-1、MMP-3mRNA表达升高更明显,有非常显著性差异(P＜0.01)。UVA照射前给予不同剂量TGF-β1处理,MMP-1、MMP-3 mRNA表达随着TGF-β1剂量增加表达降低,呈剂量依赖关系。MMP-1 mRNA表达,在大、中剂量组明显低于UVA照射组,有非常显著性差异(P＜0.01);MMP-3 mRNA表达,大剂量组明显低于UVA照射组,有非常显著性差异(P＜0.01),中剂量组与UVA照射组比较有显著性差异(P＜0.05)。不同剂量UVA照射后,各照射组Smad3表达降低于对照组,UVA 10 J/cm~2、UVA 20 J/cm~2照射组Smad3表达与对照组比较,有非常显著性差异(P＜0.01),5J/cm~2照射剂量组与对照组比较,有显著性差异(P＜0.05)。UVA照射前给予不同剂量TGF-β1处理,随着TGF-β1剂量增加,Smad3 mRNA表达升高,呈剂量依赖关系。大、中剂量组Smad3mRNA升高较明显,与UVA照射组比较,有非常显著性差异(P＜0.01)。小剂量组与UVA照射组比较,有显著性差异(P＜0.05)。3.培养皮肤成纤维细胞经UVA360nm照射,UVA累积剂量为UVA60J/cm~2后发生mtDNA 4977 bp缺失,UVA90J/cm~2时缺失明显(P＜0.01)。TGF-β1处理后,大剂量组mtDNA 4977 bp缺失减轻,与UVA照射组比较,有非常显著性差异(P＜0.01),中、小剂量组与UVA照射组比较无显著性差异(P＞0.05)。4.不同剂量UVA照射能呈现出剂量依赖的抑制HSP70表达,与对照组比较,UVA 10 J/cm~2,UVA 20 J/cm~2照射组,HSP70含量明显降低,有显著性差异(P＜0.05,P＜0.01),UVA 20 J/cm~2照射组抑制作用更明显,与对照组比较,有非常显著性差异(P＜0.01)。照射前给予TGF-β1处理后,HSP70含量随着TGF-β1剂量增加而升高,呈剂量依赖关系。大剂量组HSP70含量明显高于UVA照射组,有非常显著性差异(P＜0.01),中剂量组HSP70含量高于UVA照射组,有显著性差异(P＜0.05)。5.UVA照射体外培养的皮肤成纤维细胞导致IGF-1、KGF分泌下降,UVA10 J/cm~2、UVA 20 J/cm~2照射组明显下降,与对照组比较,有非常显著性差异(P＜0.01)。VEGF分泌升高,UVA 20 J/cm~2照射组与对照组比较,有非常显著性差异(P＜0.01)。TGF-β1处理后,TGF-β1剂量依赖性地提高UVA照射体外培养的皮肤成纤维细胞分泌三种因子的水平。大剂量组IGF-1、KGF、VEGF含量明显升高,与UVA照射组比较,有非常显著性差异((P＜0.01)。结论:1.UVA照射体外培养的皮肤成纤维细胞,导致细胞衰老,增殖活性下降;对Ⅰ型、Ⅲ型胶原合成有抑制作用,降低TGF-β1蛋白含量和mRNA表达水平,随UVA照射剂量增加而下降。提示UVA照射对胶原合成抑制作用与TGF-β1低表达有关,TGF-β1低表达和TGF-β/Smad信号传导通路受影响有关,在UVA照射导致的皮肤光老化发生过程中有着重要的作用。2.在UVA照射前加入TGF-β1,能提高体外培养皮肤成纤维细胞增殖活性,以剂量依赖方式促进UVA照射成纤维细胞Ⅰ型、Ⅲ型胶原的合成和mRNA表达。UVA照射成纤维细胞后,诱导MMP-1、MMP-3mRNA高表达,Smad3低表达;TGF-β1能降低MMP-1、MMP-3mRNA表达,增强Smad3表达,对UVA照射成纤维细胞光老化有保护作用。作用机制可能是TGF-β1促进成纤维细胞分裂增殖,TpR-Ⅱ表达增加,增强了TpR-Ⅱ对TGF-β的效应能力,促进成纤维细胞胶原合成,并通过TGF-β/Smad途径抑制MMP-1、MMP-3mRNA高表达。3.培养皮肤成纤维细胞经UVA360nm照射,UVA累积剂量为UVA60J/cm~2后发生mtDNA 4977 bp缺失,UVA90J/cm~2时缺失明显。给予一定剂量(10ng/ml)的TGF-β1后,可以使皮肤成纤维细胞mtDNA4977 bp缺失减轻。4.UVA照射抑制成纤维细胞对HSP70的表达,TGF-β1能使UVA照射成纤维细胞内HSP70表达升高,TGF-β1诱导UVA照射成纤维细胞内的HSP70高表达,对UVA照射成纤维细胞有保护作用。5.TGF-β1可提高UVA照射体外培养的皮肤成纤维细胞分泌IGF-1、KGF、VEGF三种细胞因子的水平,有利于真皮内结构的恢复和重建,有潜在的临床应用价值。更多还原

【Abstract】 BackgroundSkin aging can be divided into two basic categories:intrinsic aging and photoaging.The factors of intrinsic aging are inevitable and the specific mechanism is undiscovered.In photoaging,the ultraviolet light plays a important role.Thus skin aging caused by being repeated exposure to sunlight is named photoaging.It is characterized with coarse,skin laxitas,deep,severe wrinkling,freckles,telangiectasia and pigmentary changes or even cancer on exposed areas such as the face,neck and forearm.Fibroblasts are the most important components of the cells in the dermal. Ultraviolet(UV)leads fibroblasts to earlier aging,decreases the synthesis of collagen on the one hand,and it increases the expression of matrix metalloproteinases and degrades extracellular matrix on the other hand.In sum,it leads to skin photoaging. According to its wavelength,ultraviolet light is classified into UVC(200 to 280 nm), UVB(280 to 320 nm)and UVA(320 to 400 nm).About 99 percent the UVA reaches the Earth’s surface,whereas more than 50 percent of UVA penetrates the epidermis and arrived at fibroblasts.Therefore,UVA is closely related to skin-ageing and has attracted worldwide attention in the public health domain.As to skin-aging mechanisms,the theory of UV irradiation producing ROS’ is universally accepted at the present.As a result,in recent years domestic and foreign scholars have done more antioxidant research against UVA irradiation’s oxygen free radicals,but so far definitive anti-aging methods have not been identified.With development of studying on the anti-aging mechanism,researchers have found that the cytokine plays a very important role in regulating skin-ageing,and have devoted more attention to cytokine’s study.However,papers and reports about cytokines’ anti-skin aging are inadequate.ObjectiveIn this study,we investigateⅠ,Ⅲcollagen synthesis and expression of TGF-β1 after UVA radiate fibroblasts in vitro,and the effects of TGF-β1 to collagen synthesis. We discuss whether or not TGF-β1 inhibits the expression of MMP-1,MMP-3 and protects mitochondrial DNA(mtDNA)deletion,and analyze the effects of TGF-β1 to heat shock protein 70’s expression.Briefly we study comprehensivly the protective effects of TGF-β1 to fibroblasts in vitro being irradiated by UVA and the effects of TGF-β1 to the secretion of IGF-1,KGF,VEGF when UVA irradiates dermal fibroblasts in vitro.Methods1.We conduct experiments by culturing fibroblasts of foreskin from young adults and using the five dynasties cells.Specifically,by dividing cells into goups and using different doses of UVA(5 J/cm~2,10J/cm~2,20J/cm~2)irradiated cells,we establish control group(0 J/cm~2)and fibroblast photo-aging model.Then we test cell proliferation activity,Ⅰ,Ⅲcollagen content in supematant by MTT, enzyme-linked immunosorbent assay(ELISA)after UVA irradiation skin fibroblasts, and TGF-β1 treatment.We also testⅠ,Ⅲcollagen mRNA and TGF-β1 mRNA expression by using semi -quantitative(RT-PCR)method.2.By adding different doses of TGF-β1 to the culture medium,small dose(0.1 ng/ ml)、medium dose(1.0 ng/ml),high dose(10.0ng/ml)and with UVA15J / cm2 irradiation,we test frstlyⅠ,Ⅲcollagen content and HSP70 expression changes in supematan using immunosorbent assay(ELISA),and secondarlyⅠ,Ⅲcollagen mRNA and MMP-1,MMP-3 mRNA,Smad3 mRNA expression changes,and finally the Mitochondrial DNA(mtDNA)4977bp deletion by RT-PCR.We use enzyme-linked immunosorbent assay(ELISA)to determine the content of IGF-1, KGF,VEGF in the supernatant.Results1.Using different doses of UVA(5J/cm~2,10J/cm~2,20J/cm~2)irradiate cultured skin fibroblasts,we have found that when UVA irradiation dose increased,the fibroblast OD values decrease.Compared with the control group,UVA 10J/cm~2, UVA 20J/cm~2 irradiated group’s OD values are significantly decreased.There is a very significant difference(p＜0.01).While increasing the dose of UVA irradiation,Ⅰcollagen’s content and mRNA expression decrease in a dose-dependent manner. Being compared with the control group,UVA 10J/cm~2,UVA 20J/cm~2 irradiated group decrease significantly(p＜0.05,P＜0.01),whereas UVA 20J/cm~2 group inhibits collagen synthesis more obviously while comparing with the control group. Comparing the two groups,there is a significant difference(P＜0.01).As toⅢ collagen’s content and mRNA expression,UVA 20J/cm~2 irradiated group is significantly reduced,comparing with the control group.There is a very significant difference(P＜0.01).TGF-β1 protein content and mRNA expression decrease when the dose of UVA irradiation is increased in a dose-dependent manner.Comparing with the control group,TGF-β1 protein content and mRNA expression in UVA 10J/ cm~2,UVA 20J/cm~2 group is significantly reduced,hence a very significant difference(P＜0.01).2.Using different doses of TGF-β1 to UVA15J/cm~2 to irradiate the fibroblasts. The experiment shows that:when increasing the dose of TGF-β1,large,medium dose group OD values are significantly higher than that of the UVA irradiation group. There is a very significant difference(P＜0.01).Ⅰ,Ⅲcollagen protein content increases with the dose of TGF-β1 increased and there is a dose-dependent manner. Comparing large,medium dose of TGF-β1 group with UVA irradiation group,there are significant differences(P＜0.01,P＜0.05).High dose group promoting collagen synthesis is more obvious and there is a very significant difference(P＜0.01).With the dose of TGF-β1 increased,Ⅰ,Ⅲcollagen mRNA expression also increase in a dose-dependent manner.Ⅰcollagen mRNA expression in the large,medium-dose group is significantly increased.Comparing with UVA irradiation group,there is a very significant difference(P＜0.01).Comparing large dose group with UVA irradiation group,Ⅲcollagen mRNA expression in the large dose group is significantly increased.There is a very significant difference(P＜0.01).After adopting different doses of UVA irradiation and comparing UVA 10J/ cm~2,UVA 20J/cm~2 irradiated group with control group,MMP-1,MMP-3 mRNA expression are significantly increased.There are significant differences(P＜0.05, P＜0.01).In UVA 20 J/cm~2 irradiated group,MMP-1,MMP-3mRNA expression increase more obviously and there is a very significant difference(P＜0.01).After TGF-β1 treatment by different doses,UVA irradiates fibroblast.MMP-1,MMP-3 mRNA expression decrease with the dose of TGF-β1 increases.There is a dose-dependent manner.Large,medium dose group’s MMP-1 mRNA expression are lower than that of the control group.There is a very significant difference(P＜0.01). Large dose group’s MMP-3 mRNA expression is lower than that of the control group. There is a significant difference(P＜0.05).After different doses of UVA irradiation, Smad3 mRNA expression is lower than that of the control group.Being compared UVA 10J/cm~2,UVA 20J/cm~2 group with the control group,there is a very significant difference(P＜0.01),while being compared 5J/cm~2 irradiation dose group with the control group,there is signifcant differences(P＜0.05).Treatment with different doses of TGF-β1 to UVA irradiated fibroblasts.With increased dose of TGF-β1,Smad3 mRNA expression increases in a dose-dependent manner.In the high and medium dose group,Smad3 mRNA expression increases more obviously,while comparing with UVA irradiation group there is a very significant difference(P＜0.01). Comparing small dose group with UVA irradiation group,the difference is significant (P＜0.05).3.When the cumulative dose of UVA arrives 60J/cm~2,fibroblasts mitochondria DNA 4977 bp deletion occures.When the cumulative dose of UVA arrives 90J/cm~2, deletion is very significant(P＜0.01).After TGF-β1 treatment,the high dose group mtDNA 4977 bp deletion reduces.While being compared medium,small dose group with that of the UVA irradiation group,there is no significant difference(P＞0.05).4.Different doses of UVA irradiation have shown a dose-dependent inhibition to the HSP70 expression.Comparing UVA 10J/cm~2,UVA 20J/cm~2 irradiated group with the control group,HSP70 are significantly reduced and there are significant differences(P＜0.05,P＜0.01).Comparing UVA 20J/cm~2 irradiated group with that of the control group,the inhibition affects are more obvious.There is a very significant difference(P＜0.01).Prior to irradiation of TGF-β1 treaments,HSP70 content increases with the dose of TGF-β1 and there is in a dose-dependent manner. High dose group HSP70 is significantly higher than that of the UVA irradiation group, there is a very significant difference(P＜0.01).Medium dose group HSP70 is higher than that of the UVA irradiation group,there is significant differences(P＜0.05).5.UVA irradiation in cultured skin fibroblasts resulted IGF-1,KGF decreased secretion,UVA 10J/cm~2,UVA 20J/cm~2 irradiated group decreases significantly, comparing with the control group.There is a significant difference(P＜0.01).VEGF secretion increases in UVA 20J/cm~2 irradiated group,comparing with the control group,hence a significant difference(P＜0.01).After TGF-β1,TGF-β1 dose-dependent increases UVA irradiation in vitro skin fibroblasts secreting three factor level.Large dose group IGF-1,KGF,VEGF are significantly higher than the UVA irradiation group whereby there is a significant difference((P＜0.01). Conclusions1.UVA irradiating on cultured skin fibroblasts leads to cell aging,inhibits proliferation activity and the typeⅠ,Ⅲcollagen synthesis.Inhibiting TGF-β1 protein content and mRNA expression increase with the dose of UVA irradiation.It suggests that UVA irradiation on cultured skin fibroblasts causing collagen synthesis decreases It is also related to lower expression of TGF-β1.Low expression of TGF-β1 and TGF-β/Smad signal transduction pathway plays an important role in the skin photoaging process.2.TGF-β1 treatment before UVA irradiation can improve skin fibroblasts proliferation activity in vitro.It is in a dose-dependent manner to promote the synthesis of collagenⅠ,Ⅲand mRNA expression on UVA irradiated fibroblasts. UVA irradiating fibroblasts can induce MMP-1,MMP-3 mRNA high expression and Smad3 mRNA low expression.TGF-β1 can lower the MMP-1,MMP-3mRNA expression and enhance the expression of Smad3 mRNA.It has protective effects onto UVA irradiation fibroblast-ageing.The mechanism maybe TGF-β1 promotes fibroblast proliferation and enhances the binding ability between TβR-Ⅱand TGF-β1. It could promote fibroblast collagen synthesis.Moreover,it inhibites MMP-1, MMP-3mRNA expression through TGF-β/ Smad pathway.3.UVA(360nm)irradiating skin fibroblasts,when the cumulative dose of UVA arrives UVA60J/cm2,mtDNA 4977 bp deletion occures,and when UVA arrives 90J/cm~2 deletion is obvious.After certain dose(10ng/ml)of TGF-β1 treatment,skin fibroblast mtDNA4977 bp deletion is reduced.4.UVA irradiating fibroblast can inhibit HSP70 expression and TGF-β1 can increase it’s expression.TGF-β1 induces the HSP70 to high expression and protects fibroblast from UVA irradiation.5.TGF-β1 can improve IGF-1,KGF,VEGF levels on UVA irradiated dermal fibroblasts.更多还原