紫外线对人皮肤成纤维细胞中Hrd1及Nrf2表达的影响

目的探讨紫外线(UV)照射对人皮肤成纤维细胞中羟甲基戊二酰辅酶A还原酶降解蛋白(Hrd)1及核因子E2相关因子(Nrf)2表达的影响及可能机制。方法共收集12份人皮肤组织标本,曝光及非曝光部位标本各6份,免疫组化检测2组Hrd1及Nrf2的表达。将体外培养人皮肤成纤维细胞分为对照组、UVA组、UVB组,照光后用Western blot法检测各组成纤维细胞中Hrd1及Nrf2的表达。应用Hrd1-siRNA转染体外培养人皮肤成纤维细胞,下调细胞中Hrd1的表达后,Western blot检测成纤维细胞Nrf2的表达。应用免疫荧光分析检测细胞中Hrd1与Nrf2在细胞中的共定位情况,应用免疫共沉淀检测体外培养人成纤维细胞中Hrd1与Nrf2是否存在内源性结合。结果临床标本实验中,曝光部位皮肤组织中Hrd1表达(0.4756±0.0785)明显高于避光部位(0.1270±0.0253,t=7.317,P<0.05),曝光部位皮肤组织中Nrf2表达(0.1190±0.0217)明显低于避光部位(0.2629±0.0263,t=7.306,P<0.05)。体外培养成纤维细胞实验中,经紫外线照射后,UVA组与对照组相比,Hrd1蛋白表达水平明显增高(t=7.227,P<0.05),Nrf2蛋白表达水平明显下降(t=6.456,P<0.05);同样,UVB组与对照组相比,Hrd1蛋白表达水平明显增高(t=2.980,P<0.05),Nrf2蛋白表达水平明显下降(t=13.52,P<0.05)。应用Hrd1-SiRNA下调体外培养人纤维细胞中Hrd1的表达后,无论是经UVA照射还是UVB照射,被下调Hrd1组细胞内Nrf2的表达较未下调组明显升高。免疫荧光分析结果显示Hrd1与Nrf2在体外培养人皮肤成纤维细胞中的均有表达,并且在细胞中存在Hrd1与Nrf2共定位。免疫共沉淀验证了Hrd1与Nrf2在体外培养人皮肤成纤维细胞中存在内源性结合。结论Hrd1与Nrf2在人皮肤成纤维细胞中存在结合,紫外线照射可通过增加细胞中Hrd1的表达从而抑制Nrf2的表达。     

The expression of matrix metalloproteinase-1 mRNA induced by ultraviolet A1 (340-400 nm) is phototherapy relevant to the glutathione (GSH) content in skin fibroblasts of systemic sclerosis

The excessive deposition of collagen in systemic sclerosis (SSc) is thought to be due to an abnormal function of fibroblasts, which may be the result of an immune dysregulation in skin. Ultraviolet A1 (UVA1) irradiation has been shown to be an effective therapy. This is thought to be due to its capacity to induce matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. In the present in vitro study, the effect of UVA1 irradiation on MMP-1 was studied using skin fibroblasts from healthy controls (n=5) and patients with systemic sclerosis (n=5). In vitro irradiation studies showed that gene expression for MMP-1 after UVA1 irradiation (p<0.05) was induced in all the fibroblasts studied, but that the induction rate was greater in SSc fibroblasts than in normal ones (p<0.05). The glutathione (GSH) level was lower in SSc fibroblasts than in controls before UVA1 irradiation. However, after UVA1 irradiation, GSH levels were increased and equivalent between normal and SSc fibroblasts. These findings indicated that the relatively stronger increase in MMP-1 expression in SSc fibroblasts was due to the lower antioxidant capacity. These data support the concept that clinical responses to UVA1 radiation are influenced by the antioxidative state of the patients' skin.     

Protective and determining factors for the overall lipid peroxidation in ultraviolet A1-irradiated fibroblasts: in vitro and in vivo investigations

BACKGROUND: Lipid peroxidation (LPO) is one major effector mechanism by which ultraviolet (UV) A contributes to photoageing and the promotion of skin cancer. It is a fingerprint of photo-oxidative stress within the skin, and is initiated by several pathways, with different reactive oxygen species (ROS) and iron ions being involved. OBJECTIVES: To elucidate factors involved in UVA1-induced LPO in human dermal fibroblasts and mouse dermis, and the role of antioxidant enzymes in protecting cells against LPO. METHODS: Using a highly sensitive high-performance liquid chromatography procedure, we measured malondialdehyde (MDA), a specific metabolic tracer molecule for LPO, to determine the overall LPO produced by a given UVA1 dose in vitro and in vivo. By using the iron chelator desferrioxamine (DFO), the hydroxyl radical scavenger dimethylsulphoxide (DMSO) and fibroblasts that specifically overexpress single antioxidant enzymes, we further indirectly assessed the protective effect of manganese superoxide dismutase (MnSOD), catalase and phospholipid hydroperoxide glutathione peroxidase (PHGPx) as well as the relative importance of different ROS and the role of transitional iron for the total amount of LPO induced by a distinct UVA dose. RESULTS: UVA1 irradiation resulted in a time- and dose-dependent increase in MDA levels in vitro, and the in vitro results were shown to have in vivo relevance. Fibroblasts incubated with DFO or DMSO produced lower levels of MDA than controls, as did fibroblasts overexpressing MnSOD, catalase or PHGPx. CONCLUSIONS: The cellular iron pool and hydroxyl radicals were the most important determining factors for the total amount of MDA produced after a given UVA1 dose, and PHGPx overexpression had the greatest protective effect against LPO.     

Licochalcone A activates Nrf2 in vitro and contributes to licorice extract‐induced lowered cutaneous oxidative stress in vivo

The retrochalcone licochalcone A (LicA) has previously been shown to possess antimicrobial and anti‐inflammatory properties. In this study, we focused on pathways responsible for the antioxidative properties of LicA. In vitro, LicA protected from oxidative stress mediated by reactive oxygen species (ROS) by activating the expression of cytoprotective phase II enzymes. LicA induced nuclear translocation of NF‐E2‐related factor 2 (Nrf2) in primary human fibroblasts and elevated the expression of the cytoprotective and anti‐inflammatory enzymes heme oxygenase 1 and glutamate–cysteine ligase modifier subunit. LicA‐treated cells displayed a higher ratio of reduced to oxidized glutathione and decreased concentrations of ROS in UVA‐irradiated human dermal fibroblasts, as well as in activated neutrophils. In vivo, ultraweak photon emission analysis of skin treated with LicA‐rich licorice extract revealed a significantly lowered UVA‐induced luminescence, indicative for a decrease in oxidative processes. We conclude from these data that topical application of licorice extract is a promising approach to induce Nrf2‐dependent cytoprotection in human skin.     

Oxidative defense in cultured human skin fibroblasts and keratinocytes from sun-exposed and non-exposed skin

Skin fibroblasts and keratinocytes cultivated from chronically light-exposed skin sites have higher levels of the protective protein ferritin than cells derived from unexposed areas of the body, suggesting an adaptive response of cells exposed to chronic external insults. In the same line, ferritin levels were always found to be 2-to 7-fold higher in epidermal keratinocytes than in the underlying dermal fibroblasts of the same person thus providing the keratinocyte with continuous protection by the higher cellular ferritin content. The activation of ferritin by oxidative stress including UVA radiation could represent an important cellular defense mechanism that operates in human skin. Following low fluences of UVA radiation (2–4×10⁵ J/m²), ferritin levels increased by 20–30% in normal adult skin fibroblasts and showed a subsequent decrease at higher UVA fluences. In contrast, skin keratinocytes were not perturbed by UVA radiation exposure except for very high fluences (1.25× 10⁶ J/m²) where slight decreases in cellular ferritin were noted in 7 of the 12 cell lines. Fibroblasts derived from light-exposed skin sites that possessed higher levels of cellular ferritin were highly protected against UVA-induced membrane damage as measured by lactate dehydrogenase release compared with fibroblasts from nonexposed body sites with lower levels of ferritin. It is clear from our results that ferritin plays an important role in protection at the cellular level in human skin cells, but the role of this putative protective protein in vivo remains to be defined.     

The lazaroid tirilazad is a new inhibitor of direct and indirect UVA-induced lipid peroxidation in human dermal fibroblasts

Lipid peroxidation caused by oxidative stress within the tissue leads to destruction and dysfunction of cellular membranes. Human dermal fibroblasts in the skin are subject to constant photooxidative stress caused mainly by deeply penetrating UVA irradiation. Therefore, the membrane damage caused by this photooxidative stress may be a major promoter of photoaging and photocarcinogenic processes initiated and promoted by long-term UVA exposure of the skin. The oxidative destruction is counterbalanced by a complex network of enzymatic and nonenzymatic antioxidants creating the skins line of defence against UVA-induced reactive oxygen species. The lazaroid tirilazad represents a new synthetic group of antioxidants with structural molecular similarity to glucocorticosteroids. We investigated the antioxidative capacity of tirilazad by determining its effects on the levels of malondialdehyde (MDA), as a marker of lipid peroxidation, induced directly or indirectly by UVA in human dermal fibroblasts. In a time- and dose-dependent kinetic, we demonstrated that fibroblasts incubated with tirilazad are well protected against subsequent UVA irradiation and show no increase in MDA levels similar to the unirradiated controls. This was also observed when lipid peroxidation was caused chemically by incubation of human dermal fibroblasts with 200 M Fe³⁺-citrate and 1 mM ascorbyl phosphate as a model of indirect UVA-induced skin damage. Lysates of fibroblasts treated this way showed a tenfold increase in MDA levels, whereas preincubation with tirilazad resulted in a significantly lower increase in MDA levels. Furthermore, in a comparison with the well-established radical scavenger Trolox, an -tocopherol analogue, tirilazad offered better protection to the membranes. Our results demonstrate for the first time that the lazaroid tirilazad is an effective inhibitor of direct and indirect UVA-induced increases in MDA as a marker of lipid peroxidation in human dermal fibroblasts.     

Interleukin-1beta and macrophage migration inhibitory factor (MIF) in dermal fibroblasts mediate UVA-induced matrix metalloproteinase-1 expression

BACKGROUND: Exposure to solar UV radiation is the main environmental factor that causes premature aging of the skin. Matrix metalloproteinases (MMP)-1 is a member of the MMP family and degrades types I and III collagens, which are the major structural components of the dermis. OBJECTIVE: We evaluated the involvement IL-1beta and macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation. METHODS: IL-1beta and MIF in MMP-1 expression in cultured human dermal fibroblasts and the UVA effects on MMPs production using IL-1alpha/beta-deficient mice were analyzed. Furthermore, fibroblasts derived from MIF-deficient mice were used to analyze the effect of IL-1beta-induced MMPs production. RESULTS: IL-1beta-enhanced MIF expression and induced MMP-1 in cultured human dermal fibroblasts. IL-1beta-induced MMP-1 expression is inhibited by neutralizing anti-MIF antibody. Dermal fibroblasts of IL-1alpha/beta-deficient mice produced significantly decreased levels of MMPs compared to wild-type mice after UVA irradiation. Furthermore, fibroblasts of MIF-deficient mice were much less sensitive to IL-1beta-induced MMPs production. On the contrary, IL-1beta produced significantly decreased levels of MMPs in MIF-deficient mice fibroblasts. The up-regulation of MMP-1 mRNA by IL-1beta stimulation was found to be inhibited by a p38 inhibitor and a JNK inhibitor. In contrast, the MEK inhibitor and inhibitor were found to have little effect on expression of MMP-1 mRNA. CONCLUSIONS: IL-1beta is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts, and IL-1beta and MIF cytokine network induce MMP-1 and contribute to the loss of interstitial collagen in skin photoaging.     

Photoprotection of bacterial-derived melanin against ultraviolet A–induced cell death and its potential application as an active sunscreen

Background  The increase in the incidence of non-melanoma skin tumours, photoaging, and immunosuppression demand for more effective sunscreen on ultraviolet A (UVA) irradiation.
Objectives  The aim of the study is to evaluate the photoprotective effects of a bacterial-derived melanin against UVA-induced damages in vitro and in vivo .
Methods  Human fibroblasts were used to assess the role of the bacterial-derived melanin on cell viability against UVA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and nuclear morphology were employed to evaluate the photoprotection at the cellular level. Fluorometric assays were performed to detect the formation of reactive oxygen species (ROS) in the cells. Evaluations of the bacterial-derived melanin as a sunscreen were measured by transmission test and persistent pigment darkening on human skin.
Results  Bacterial-derived melanin efficiently scavenged ROS in the fibroblasts after UVA irradiation. The cell viability of xeroderma pigmentosum (XP) fibroblast treated with varied doses of melanin increased dramatically in comparison with untreated control and the treated XP fibroblasts became more resistant to UVA-induced apoptosis than normal fibroblasts. Although the relative transmission didn't change too much with different concentration of bacterial-derived melanin, this melanin could keep UVA-irradiated skin from pigment darkening and act as an active sunscreen on skin.
Conclusions  The bacterial-derived melanin provided significant protection to fibroblast cell and human skin against the UVA radiation. It has the potential to be developed as an active sunscreen for the patients with photosensitivity skin to sun exposure.     

Ultraviolet-A radiation induces adhesion molecule expression on human dermal microvascular endothelial cells

Ultraviolet radiation is capable of inducing numerous skin reactions. Considerable amounts of UVA radiation penetrate the epidermis and reach the microvascular endothelium of the papillary dermis. In order to investigate putative direct effects of UV radiation on endothelial cells, we studied adhesion molecule expression by immunostaining procedures and FACS analysis, following irradiation of normal human skin and cultured human dermal endothelial cells. Enhanced immunostaining for ICAM-1 and E-selectin was detected in biopsies taken after in vivo UVA and UVB irradiation, compared with non-irradiated control skin. On cultured human dermal endothelial cells, however, ICAM-1 and E-selectin were inducible by UVA but not UVB. The induction was dose-dependent, peaking at 20 J/cm² for both adhesion molecules, and time-dependent, peaking after 6 and 24 h for E-selectin and ICAM-1, respectively. Expression of VCAM-1 and PECAM/EndoCAM/CD31 was unaffected by any UV-radiation modality. The functional integrity of irradiated cells was monitored by an exclusion assay of the fluorescent dye 7-AAD, and by staining for the cytoskeletal proteins actin and vimentin. Our results demonstrate that dermal microvascular endothelial cells are a critical and direct target of UVA, and suggest they may play a pivotal role in UV-induced inflammatory skin conditions.     

UVA1 irradiation induces deoxyribonuclease dependent apoptosis in cutaneous T-cell lymphoma in vivo

Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature T-cells, predominantly of the helper phenotype, that primarily invade the skin. Different photo- and chemotherapeutic treatments are known to be beneficial in early-stage CTCL. This observation has initiated prospective investigations into the efficacy of phototherapeutic regimens. The purpose of our study was to investigate the ability of medium-dose UVA1 phototherapy (60 J/cm2) to induce apoptosis (programmed cell death) in skin infiltrating T-cells of CTCL in vivo. We describe the results of three different staining methods for formalin-fixed, paraffin-embedded tissue sections. The in situ end-labeling (ISEL) procedure, nuclear staining using the DNA-binding fluorochrome Hoechst 33342, and immunohistochemistry using polyclonal antibodies against recombinant mouse deoxyribonuclease I (DNase I) demonstrated that UVA1 irradiation was able to induce marked apoptosis in CTCL. Thereby, ISEL and Hoechst staining clearly revealed DNA-condensation and nuclear fragmentation, accompanied by the formation of typical "apoptotic bodies". The accumulation of DNase I immunoreactivity in the cytoplasm of lymphocytes in UVA1 irradiated skin indicated that DNase I or DNase I-related endonucleases may have acted as apoptotic endonuclease(s) which were synthesized after UVA1 irradiation prior to their apoptotic elimination.     

Accumulation of elafin in actinic elastosis of sun-damaged skin: elafin binds to elastin and prevents elastolytic degradation

Elafin has a primary structure with two functional domains; a transglutaminase substrate domain at the N-terminus and a protease inhibitor domain at the C-terminus. Elafin expression has so far been reported only for epithelial tissues. Accumulation of elafin was immunohistochemically detected in the actinic elastosis of sun-damaged skin. Exposure of normal skin to UVA induced elafin expression that colocalized with elastic fibers. Incubation of synthetic transglutaminase substrate domain of elafin and elastin molecules in the presence of tissue transglutaminase in vitro resulted in the formation of a higher molecular complex on SDS-PAGE. Elafin expression was not detected in normal cultured skin fibroblasts, but was induced by UVA irradiation at both messenger RNA and protein levels. When radiolabeled insoluble elastin was incubated with recombinant full-length elafin and tissue transglutaminase, insoluble elastin became more resistant to neutrophil elastase digestion. These results indicate that (1) dermal fibroblasts potentially express elafin on UV irradiation, (2) UV-mediated elafin interacts with elastin, and (3) the elafin-elastin complex protects elastic fibers from elastolytic degradation, leading to the accumulation of elastic fibers in the actinic elastosis of sun-damaged skin. The transglutaminase substrate moiety of elafin plays an important role in anchoring elafin at its proper sites of action during UV-induced aging processes.     

Hepatocyte growth factor establishes autocrine and paracrine feedback loops for the protection of skin cells after UV irradiation

Hepatocyte growth factor (HGF) is a multifunctional cytokine, which, among various other activities, acts as a growth factor for melanocytes and has recently been implicated in the pathogenesis of malignant melanoma. In the skin, the main source for HGF is dermal fibroblasts (FB). Here, we have investigated the regulation of HGF production and secretion by cytokines derived from UV-irradiated keratinocytes (KC) and by direct UV irradiation. We demonstrate that supernatants of ultraviolet (UV)B-irradiated KC strongly induce HGF production in FB, and that this effect was mediated primarily by IL-1alpha. Direct irradiation of FB with UVB had no effect on HGF expression. In contrast, irradiation with UVA1 strongly upregulated HGF mRNA production and secretion of the functional protein. Addition of neutralizing anti-HGF antibodies after UVA1 irradiation, as well as transfection of FB with HGF small-interfering RNA (siRNA); which completely abrogated HGF secretion led to a dramatic rise of FB apoptosis demonstrating that autocrine HGF efficiently protected FB from UVA1-induced apoptosis. Our data suggest that upregulation of HGF plays a role in skin homeostasis after UV irradiation. However, a negative side effect of UV-induced HGF secretion by dermal FB might represent a decisive factor for induction and/or progression of melanoma.     

circIGF2BP3调控光老化皮肤成纤维细胞自噬水平的研究

【摘要】 目的 初步探究circIGF2BP3对光老化皮肤成纤维细胞自噬水平的影响。方法 取中山大学附属第三医院泌尿外科6例儿童包皮环切术后包皮组织,分离培养成纤维细胞,以每日10 J/cm2长波紫外线（UVA）连续照射14 d,建立UVA诱导的光老化成纤维细胞模型（UVA处理组）,未经处理的正常成纤维细胞作为对照组,β半乳糖苷酶染色、Western印迹法检测P21蛋白表达,CCK8法检测细胞活力验证建模是否成功。Western印迹法检测光老化成纤维细胞中自噬相关蛋白P62、LC3-Ⅱ、LC3-Ⅰ表达,qRT-PCR验证光老化与正常成纤维细胞间circIGF2BP3表达差异,并对其进行生物学注释。将原代成纤维细胞分为4组：空载组、UVA + 空载组,circIGF2BP3过表达组、UVA + circIGF2BP3过表达组,Western印迹法检测各组细胞中自噬相关蛋白表达水平。两独立样本均数的比较采用t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果 UVA处理组β半乳糖苷酶染色阳性率（61.33% ± 5.78%）、P21蛋白表达（1.25 ± 0.03）均显著高于对照组（6.37% ± 0.32%、1.00 ± 0.05,t = 9.49、4.26,P < 0.01、< 0.05）,而细胞活力（74.33% ± 3.48%）显著低于对照组（100%,t = 7.38,P < 0.01）。UVA处理组P62蛋白表达及LC3-Ⅱ/Ⅰ比值均显著高于对照组（均P < 0.05）。光老化成纤维细胞中circIGF2BP3的相对表达量为0.72 ± 0.04,显著低于对照组（1.00 ± 0.03）,t = 5.46,P < 0.01。circIGF2BP3过表达组P62蛋白表达（0.60 ± 0.01）及LC3-Ⅱ/Ⅰ比值（0.71 ± 0.01）均显著低于空载组（1.00 ± 0.02、1.00 ± 0.01;t = 16.25、2.75,P < 0.01、P < 0.05）;UVA + circIGF2BP3过表达组P62蛋白表达（1.05 ± 0.02）及LC3-Ⅱ/Ⅰ比值（2.04 ± 0.05）亦均显著低于UVA + 空载组（1.31 ± 0.02、2.72 ± 0.14;t = 10.49、6.47,均P < 0.01）。结论 circIGF2BP3可调控UVA诱导的光老化皮肤成纤维细胞自噬水平,为防治光老化提供了新的潜在治疗靶点。     

Immunoprotective UVA (320-400 nm) irradiation upregulates heme oxygenase-1 in the dermis and epidermis of hairless mouse skin

The induction of heme oxygenase-1 (HO-1) by ultraviolet A (UVA) (320-400 nm) radiation provides a protective cellular defence against oxidative stress, and has been well demonstrated in cultured human skin fibroblasts, although keratinocytes were unreactive. The UVA responsiveness of HO-1 however, has not been confirmed in intact skin. Previously, we reported that UVA-inducible HO enzyme activity in mouse skin is protective against UVB-induced immunosuppression. This study identifies the induced HO isoform and its localization in mouse skin irradiated in vivo with such an immunoprotective UVA dose. We found that HO-1 mRNA was expressed in UVA-irradiated skin, but not in normal or UVB-irradiated skin, whereas constitutive HO-2 was always present. UVA-irradiated skin had increased HO enzyme activity and bilirubin content, and decreased heme content, consistent with HO-1 induction. In situ hybridization and immunohistochemical staining localized HO-1 mRNA and protein to both epidermis and dermis, with strongest expression in basal keratinocytes and weaker expression in dermal fibroblast-like and other cells, in contrast with UVA-induced HO-1 in cultured human skin fibroblasts. This suggests that cultured skin cells may not fully represent skin functions in vivo, or that there may be inherent differences between human and hairless mouse skin HO-1 responses.     

Immunoprotective haem oxygenase induction by ultraviolet A (320-400 nm) radiation in the mouse is inhibited in interferon-gamma null mice

BACKGROUND: A protective role for the ultraviolet (UV) A waveband against immunosuppression induced by UVB (280-320 nm) radiation has been identified. The mechanism for UVA immunoprotection was found to involve two apparently unrelated mediators, the T-helper-1-associated proinflammatory cytokine interferon (IFN)-gamma and the UVA-induced redox-regulated stress protein, haem oxygenase (HO). OBJECTIVES: To identify a relationship between these two immune regulators. METHODS: The HO response to UVA radiation in the skin and liver was examined in mice with a targeted disruption of the IFN-gamma gene, known to be unresponsive to UVA photoimmunoprotection. Results IFN-gamma null mice did not respond to UVA irradiation with the normal upregulation of HO activity in either the irradiated skin or the liver. Injection of these mice with recombinant IFN-gamma previously shown to restore the UVA-photoimmunoprotective effect, here partially and dose-responsively restored their ability for induction of HO activity in both skin and liver following UVA irradiation. CONCLUSIONS: IFN-gamma appears to be a prerequisite for the immunoprotective induction of HO, although other mediators may also be involved. The UVA responsiveness of HO in an internal organ such as the liver suggests the existence of a soluble UVA-induced mediator from the skin, which may be IFN-gamma.     

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。