咖啡因可保护UVB致人皮肤成纤维细胞的损伤

目的：确定咖啡因对中波紫外线（UVB）照射致体外培养人皮肤成纤维细胞氧化损伤的防护作用.方法：分离培养人成纤维细胞,分为空白对照组、咖啡因组、UVB照射组、UVB照射＋咖啡因组,UVB照射剂量为30 mJ/cm2.用MTT法检测细胞增殖活性,酶生化比色法检测细胞丙二醛（MDA）含量、超氧化物歧化酶（SOD）及谷胱甘肽过氧化物酶（GSH-Px）活性.结果：UVB照射前后用咖啡因处理能使成纤维细胞存活率提高、MDA产生减少,酶的活性增强.结论：咖啡因对UVB照射损伤成纤维细胞具有一定保护性作用,其机制可能与抑制氧化损伤和增强抗氧化能力有关.     

UVB对皮肤成纤维细胞相关骨架蛋白表达的影响

目的:确定UVB对皮肤成纤维细胞骨架蛋白的影响.方法:用150 mJ/cm2的UVB照射培养的皮肤成纤维细胞,用MTT法检测细胞活性,用Western blot法检测α-微管蛋白、β-微管蛋白、β-肌动蛋白和波形蛋白的含量.结果:150 mJ/cm2 UVB照射后β-微管蛋白含量逐渐降低,波形蛋白逐渐升高,α-微管蛋白和β-肌动蛋白改变不明显.结论:在UVB诱导人皮肤成纤维细胞光损伤过程中,β-微管蛋白和波形蛋白可能发挥着重要作用.     

UVB照射诱导皮肤成纤维细胞早期衰老的研究

目的 探讨UVB诱导的细胞衰老与肿瘤发生的关系。方法 噻唑蓝（MTT）法检测UVB辐射后的细胞增殖情况,筛选诱导衰老适宜的亚毒性剂量和照射次数。染色法检测衰老相关的β-半乳糖苷酶（SA β-gal）活性。RT－PCR检测衰老相关基因纤维结合素（FN）、骨结合素（ON）和平滑肌22（SM22）的表达。结果 以10 mJ/cm2的亚毒性剂量连续5次照射人成纤维细胞后,衰老的生物学特征得以明显表现：①MTT法检测显示细胞增生能力的减弱。②照射组具有SA β-gal活性的阳性细胞明显增加,照射组和对照组的阳性率分别为82.0%和33.7%（P < 0.01）。③3种衰老相关基因FN、ON和SM22的表达亦明显增强,分别约为对照组细胞的2.7、2.0、2.3倍（P < 0.05）。结论 反复亚毒性剂量UVB照射人成纤维细胞,初步建立一种UVB诱导的应激诱导的早期衰老（SIPS）模型。     

人参皂苷Rg1对UVB损伤PG12及成纤维细胞的保护作用

目的研究人参皂苷Rg1对中波紫外线（UVB）损伤皮肤成纤维细胞以及作为皮肤神经细胞模型的PC12细胞的保护作用。方法实验分为UVB模型组、UVB＋Rg1三个不同浓度保护组、对照组,分别以60mJ／cm^2、100mJ／cm^2强度UVB造成培养的皮肤成纤维细胞、皮肤神经细胞模型PC12（神经元化）细胞损伤,用MTT法检测细胞增殖活性,酶生化法检测光损伤前后细胞培养上清SOD活性、MDA含量。结果强度为60mJ／cm^2、100mJ／cm^2的中波紫外线分别可以造成体外培养的人皮肤成纤维细胞、神经元化PC12细胞增殖活性降低,细胞培养上清SOD活性降低,MDA含量升高,与UVB模型组相比均P〈0．05。给定浓度范围的Rg1能增加细胞的增殖活性,增加细胞培养上清SOD活性、降低MDA含量。结论紫外线可以造成体外培养的人皮肤成纤维细胞及PC12细胞氧化损伤,人参皂苷Rg1对损伤的细胞具有一定保护作用,其机制可能与它的抗氧化、增强细胞活力有关     

紫外线对人皮肤成纤维细胞的影响

紫外线辐射可造成皮肤损伤,引起皮肤出现红斑、光老化等。紫外线作用于成纤维细胞,引起细胞因子分泌及基因表达的改变,不仅能造成真皮层的损伤,还可被用来治疗某些疾病,如局限性硬皮病。从紫外线对皮肤成纤维细胞的损伤、成纤维细胞对紫外线的防御反应及紫外线的应用等方面阐述了紫外线对成纤维细胞的影响。     

长波紫外线照射对皮肤成纤维细胞产生白三烯B4及5-脂氧合酶mRNA表达的影响

紫外线照射可以使皮肤细胞释放多种炎症介质,从而引起皮肤产生急性或慢性炎症反应.长波紫外线(UVA)具有较强的穿透力,可穿过表皮到达真皮上部,主要作用于皮肤成纤维细胞,是引起皮肤炎症反应和皮肤光老化的重要因素.     

白藜芦醇对UVB照射的人皮肤成纤维细胞MMP-1水平及细胞凋亡的影响

目的初步探寻红葡萄酒中主要活性成分白藜芦醇对紫外线照射皮肤保护作用的可能机制。方法白藜芦醇对UVB照射的人皮肤成纤维细胞基质金属蛋白酶(MMP)-1水平及细胞凋亡的影响。将培养的人皮肤成纤维细胞分为5组,A组:无UVB照射无干预组,B组:UVB照射无干预组,C组:UVB照射1μmol/L白藜芦醇干预组,D组:UVB照射10μmol/L白藜芦醇干预组,E组:UVB照射100μmol/L白藜芦醇干预组。UVB照射剂量均为30mJ/cm2。用免疫组织化学方法分别检测5组细胞中MMP-1水平,用磷脂酰丝氨酸外翻分析(AnnexinV法)检测细胞凋亡率。结果 A～E组5组细胞MMP-1阳性细胞评分及凋亡率差异均有统计学意义(P均<0.05)。A组评分及凋亡率均明显低于B组,两组比较差异有统计学意义(P均<0.05),B组与C,D,E组评分及凋亡率比较差异均有统计学意义(P均<0.05),C～E组3组评分及凋亡率比较差异亦有统计学意义(P<0.05)。结论白藜芦醇可以抑制UVB照射诱导的人皮肤成纤维细胞MMP-1水平的升高,并可减少细胞因UVB照射引起的凋亡,白藜芦醇可能通过此机制对紫外线照射皮肤起到保护作用。     

UVA照射对皮肤成纤维细胞组织蛋白酶K表达的影响

目的 探讨皮肤成纤维细胞中组织蛋白酶K(Cathepsin K,CatK)的表达随UVA照射后时间及其剂量的变化.方法 原代培养的皮肤成纤维细胞来自儿童包皮,10代以内的细胞行后续实验.先以10 J/cm2 UVA照射皮肤成纤维细胞,在照射后24、48、72 h提取照射组和对照组细胞蛋白和mRNA.再以10 J/cm2、20 J/cm2、30 J/cm2 UVA照射皮肤成纤维细胞,在照射后48 h收集细胞.用RT-PCR和Western印迹法分别检测各组间CatK mRNA、蛋白的表达的差异.结果 10 J/cm2 UVA照射后1、2、3d,照射组CatKmRNA表达分别为0.351±0.038(对照组0.177±0.006)、0.510±0.017(对照组0.176±0.002)、0.313±0.012(对照组0.173±0.002).照射组CatK蛋白灰度值分别为1.76±0.27(对照组0.82±0.45)、2.97±0.36(对照组1.58±0.15)、2.23±0.14(对照组1.29±0.32),照射组CatKmRNA、蛋白表达均较对照组升高(P＜0.05),且以照射后第2天表达最高.10、20、30 J/cm2UVA照射后,48 h皮肤成纤维细胞中CatK mRNA表达分别为对照组的2.34、2.91、3.18倍,CatK蛋白表达分别为对照组的1.77、2.82、3.64倍,均随UVA剂量的升高而增加,其差异有统计学意义(P＜0.05).结论 CatK在UVA照射后皮肤成纤维细胞中的表达升高.     

紫外线照射对成纤维细胞染色体端粒DNA复制的影响

目的:旨在DNA水平探讨紫外线启动人皮肤光老化的机制,以加深对光老化的理解,并为减缓光老化进程提供新靶点.方法:紫外线照射原代人成纤维细胞,用比色法测定成纤维细胞的增殖活性;用1 000 mJ/cm2紫外线照射细胞,采用实时定量PCR方法测定照射与未照射细胞不同代数的端粒长度;不同剂量紫外线照射同代细胞,并测定端粒长度.结果:紫外线照射与未照射细胞的端粒长度均有随着细胞的复制传代而逐渐缩短趋势;在不同剂量紫外线照射的同一代细胞之间,紫外线剂量越高,端粒长度则越短,高剂量组与未照射组相比有统计学差异(P<0.05).结论:单次大剂量紫外线照射可以使端粒长度变短.急性光损伤可能启动光老化的早期进程.     

UVA1对人工皮肤成纤维细胞光损伤的影响

目的探讨不同UVA1照射剂量对人工皮肤真皮细胞形态及细胞活性的影响,为建立人工皮肤光损伤模型提供依据。方法以不同剂量UVA1（0J／cm^2-80J／cm^2）照射人工皮肤,通过HE染色,从形态学上观察经UVA1照射后真皮成纤维细胞数量的变化,通过MTT法检测真皮成纤维细胞的活性。结果HE染色示真皮成纤维细胞的数量随UVA1剂量的增大而减少,于真皮浅层较为明显;MTT法示与0J／cm^2组相比,20J／cm^2和30J／cm^2UVA1照射人工皮肤时成纤维细胞活性未受到明显的抑制（P〉0．05）．UVA1剂量大于40J／cm^2时细胞活性下降明显（P〈0．05,P〈0．001）,呈剂量依赖性。结论UVA1照射剂量大于40J／cm^2是造成人工皮肤真皮成纤维细胞光损伤的始剂量。     

UVB对人皮肤成纤维细胞自噬影响的初步研究

目的:观察不同照射剂量及不同时间点的UVB照射对人皮肤成纤维细胞自噬功能的影响.方法:分别用不同照射剂量的UVB照射培养的人皮肤成纤维细胞后,用MDC法观察各照射组成纤维细胞的形态及自噬体阳性细胞的数量.同时将照射后的成纤维细胞用MDC法观察各时间点自噬体阳性细胞数量.结果:0、100、200、300、400 mJ/cm2各组自噬体阳性细胞百分数分别为5.975±1.889,21.400±4.286,89.550±4.282,45.500±9.423,36.250±9.996.各组之间除300 mJ/cm2与400 mJ/cm2组之间无显著性差异(P＞0.05)外,其余均存在显著性差异(P<0.05).12、24和48 h自噬体阳性细胞百分比分别为89.550±4.282、50.500±7.328和14.575±4.104.各组之间均存在显著性差异(P＜0.01).结论:UVB在中小照射剂量时可以上调人皮肤成纤维细胞自噬,而进一步加大照射剂量则呈下降趋势.UVB对人皮肤成纤维细胞自噬的影响随时间推移逐渐弱化.     

Caspase-3在UVB诱导人皮肤成纤维细胞凋亡中的作用

目的：评价caspase-3在UVB诱导皮肤成纤维细胞凋亡中的作用。方法：皮肤成纤维细胞经150mJ／cm2 UVB照射后,用MTF法检测细胞活性,用Hoechst33258染色法检测细胞凋亡,用抑制剂Z-DEVD-FMK抑制caspase-3活性后,检测凋亡细胞数量。结果：UVB明显抑制成纤维细胞的活性,并导致细胞凋亡,并呈时间-效应关系;加入抑制剂Z-DEVD-FMK抑制了UVB导致的细胞凋亡。结论：Caspase-3在UVB照射诱导皮肤成纤维细胞凋亡中发挥重要作用。     

Influence of human dermal fibroblasts on epidermalization

Using a method that allowed the reconstruction of simplified living human skin in vitro, we investigated the effects of collagen texture and dermal fibroblasts on epidermal growth. Like in vivo skin, our in vitro model comprised two tissues: a dermal equivalent and an overlying epidermis. It permitted measurement of epidermal growth and therefore evaluation of the effect of the dermal equivalent on this growth. Epidermal growth was enhanced when the collagen matrix had previously been reorganized by fibroblasts, and was greatest when living fibroblasts persisted in this matrix. On cell-free collagen gel and on collagen matrices containing dead fibroblasts, epidermal growth increased when the medium was conditioned by fibroblasts grown in monolayers. We conclude that the function of the fibroblasts is not only to synthesize and degrade the extracellular matrix, but also to regulate epidermalization; on the one hand by remodeling the collagen fibers, and on the other by secreting diffusible factors that promote epidermal growth. These results underline the importance of fibroblasts in dermo-epidermal interactions, and show that the skin equivalent culture model provides a way to quantitatively study these interactions.     

Effects of human neutrophil peptide-1 on the expression of interstitial collagenase and type I collagen in human dermal fibroblasts

Human neutrophil peptide-1 (HNP-1), a defensin with antimicrobial properties, is also thought to promote wound healing. To elucidate the mechanism by which wound healing is facilitated by this factor, we investigated the effect of HNP-1 on the expression of interstitial collagenase (matrix metalloproteinase 1, MMP-1), collagen types I and III, and tissue inhibitor of metalloproteinase 1 (TIMP-1) by cultured fibroblasts by means of RT-PCR and ELISA. Our results showed that synthetic HNP-1 increased the expression of proalpha1(I) collagen mRNA and protein. In contrast, the expression of MMP-1 was decreased at both the mRNA and protein levels. Our observations suggest that HNP-1 may promote wound repair by enhancing extracellular matrix deposition and by controlling its degradation.     

SIRT1 modulates expression of matrix metalloproteinases in human dermal fibroblasts

Background SIRT1, an NAD⁺‐dependent histone/protein deacetylase, controls a broad range of cellular functions. Objectives We examined if SIRT1 is involved in the regulation of matrix metalloproteinase (MMP) expression in human dermal fibroblasts. Methods We studied the effect of inhibition of SIRT1 by specific inhibitor and small interfering RNA (siRNA) on MMP‐1 and MMP‐3 expression in human dermal fibroblasts. Results Treatment with a potent and selective inhibitor of SIRT1, EX‐527, increased the basal expression levels of MMP‐1 and MMP‐3 proteins. Knockdown of endogenous SIRT1 by siRNA led to increased expression of MMP‐1 and MMP‐3 at both mRNA and protein levels. SIRT1 knockdown also upregulated MMP protein induction caused by an inflammatory cytokine, interleukin (IL)‐1β. Moreover, treatment with a SIRT1 activator, resveratrol, significantly suppressed IL‐1β‐mediated induction of MMP‐1, which was attenuated by pretreatment with EX‐527. Finally, MMP‐1 promoter activity was increased by EX‐527 in cells treated with or without IL‐1β. Conclusions Our findings suggest that SIRT1 exerts a negative regulatory role in the production of MMP‐1 and MMP‐3 in human dermal fibroblasts.     

Effects of oestrogen agonists on human dermal fibroblasts in an in vitro wounding assay

Abstract:  Oestrogen and dehydroepiandrosterone (DHEA) improve wound healing, but circulating levels decline significantly with age. Recently, the selective oestrogen receptor modulators (SERMs) tamoxifen and raloxifene have been shown to improve age-associated impaired wound healing. Therefore, we have evaluated the effects of 17β-oestradiol, ERα and ERβ agonists, tamoxifen, raloxifene and DHEA on human dermal fibroblasts using an in vitro wound assay. An ERα agonist, 17β-oestradiol and DHEA all significantly accelerated cell migration; the DHEA effect was blocked with an aromatase inhibitor. Tamoxifen, raloxifene and DHEA all significantly increased DNA synthesis; the DHEA stimulatory effect was reversed by an aromatase inhibitor. This study demonstrates that 17β-oestradiol, an ERα agonist, tamoxifen, raloxifene and DHEA (following conversion to oestrogen) all have significant effects on human fibroblasts, the key mesenchymal cell involved in the wound healing process. Further understanding of the mechanisms involved may have important implications for the management of age-related impaired wound healing.