NB-UVB对黑素细胞黑素合成相关基因表达的影响

目的:研究NB-UVB照射对黑素细胞的细胞活性、黑素合成、黑素合成相关基因表达的影响.方法:培养黑素细胞系PIG1,利用MTT法检测不同剂量NB-UVB(400、800、1200、1600、2000 mJ/cm2)照射后细胞的增殖率,采用NaOH裂解法测定细胞中的黑素含量,RT-PCR和Western blot方法检测...     

UVA1 impairs the repair of UVB‐induced DNA damage in normal human melanocytes

The exact correlation between melanoma and sun‐light is still a controversially debated issue. Although natural sunlight contains various ratios of UVA and UVB, most investigators so far focused on the effects of single solar wavebands and neglected possible interactions. Therefore, in this study primary human melanocytes of three donors were simultaneously exposed to physiologic doses of UVA1 and UVB. Effects on apoptosis were analysed using annexin V assays and cell death ELISAs, and effects on DNA damage were investigated using southwestern slot blots. While UVA1 did not influence UVB‐induced apoptosis, UVA1 impaired the repair of UVB‐induced cyclobutane pyrimidine dimers (CPD) as the amount of CPD was 1.8 times higher in UVA1 + UVB than in UVB only exposed melanocytes six hours after irradiation. We conclude that UVA1 might contribute to melanomagenesis as it partially inhibits the repair of UVB‐induced CPD in human melanocytes while it does not affect UVB‐mediated apoptosis.     

Placental extracts regulate melanin synthesis in normal human melanocytes with alterations of mitochondrial respiration

Placental extracts have been widely used as skin lightening agents in the Japanese cosmetic market. Here, we show that placental extracts contain factors that can decrease or increase melanin synthesis by normal human melanocytes in vitro in possible association with mitochondrial respiration. When normal human melanocytes were treated with a whole porcine placental extract, melanin synthesis was decreased. In contrast, a porcine placental extract in which exudates and insoluble materials including lipids had been removed increased melanin synthesis. In addition, the amount of tyrosinase, the enzyme critical for melanin synthesis, changed in accordance with the alteration of melanin synthesis. Interestingly, the amount of manganese‐dependent superoxide dismutase (MnSOD), a mitochondrial‐resident antioxidant enzyme, was increased when melanin synthesis was decreased by the whole placental extract. Mitochondrial respiration and glycolysis also changed following treatment with the placental extracts. These results suggest that placental extracts contain factors that can increase or decrease melanin synthesis by normal human melanocytes and that mitochondrial function may be associated with the placental extract‐induced regulation of melanogenesis.     

Effects of various doses of glutathione on the proliferation,viability, migration,and ultrastructure of cultured human melanocytes

Normal human cultured melanocytes were exposed to various glutathione concentrations (0.1, 0.5, 1.0, 5.0, and 10.0 mg/mL) for 72 hours. At the end of the experiment, proliferation, viability, migration, and ultrastructural changes were monitored. Glutathione at the doses of 0.5 to 10.0 mg/mL reduced the viability of melanocytes significantly as compared to the control (P < .05). Glutathione significantly reduced the proliferation of melanocytes at the doses of 0.5 to 10.0 mg/mL as compared to the control (P < .001). Glutathione at 0.5 to 10.0 mg/mL significantly reduced the migration of melanocytes as compared to the control (P < .001). The percentage of mature melanosomes was 53.43% in control and 50.58%, 41.83%, 33.4%, and 8.95% in 0.1, 0.5, 1.0, and 5.0 mg/mL glutathione exposed cells, respectively. This reduction in the number of mature melanosomes was statistically significant as compared to the control. However, no cytotoxic effects were recognized by electron micrographs. These results encourage the potential implementation of glutathione as a skin‐lightening agent. However, this study is limited by cell culture and ultrastructural. It should therefore be expanded in the future to include patients with pigmentary disorders.     

Effect of antioxidants and free radical scavengers on protection of human skin against UVB, UVA and IR irradiation

Background/aims: This study was designed to observe the effect of a mix of commercial antioxidants and free radical scavengers (AO) on protection of human skin against different wavelengths of the solar spectrum: 280-320 nm (UVB), 320-400 nm (UVA) and 400-900 nm (IR).
Methods: Healthy volunteers were chosen from the local population and exposed to the three aspects of the solar spectrum. Erythema at 24 h, immediate pigment darkening (IPD) and immediate erythema were employed as markers for UVB, UVA and IR exposure, respectively.
Results: Based on 24 h erythema measurements, the AO mix afforded about 2.6-fold (163%) protection against UVB-induced erythema (Table 1). IPD measurements after UVA exposure exhibited 76% protection afforded by the AO mix. The AO mix reduced IR-induced increases in erythema and blood flow by 43% and 52%, respectively (Table 1).
Conclusions: Data from these studies suggest that treatment with antioxidants prior to exposure to solar irradiation allows protection of human skin against IR as well as UVB and UVA. Antioxidants and free radical scavengers may be valuable adjuncts to traditional sunscreens, for protection of skin against the effects of actinic exposure that, over a long term, can result in formidable consequences such as skin cancer and photo-aging.     

Effect of UVB 311 nm irradiation on normal human skin

Ultraviolet radiation B (UVB) on the skin induces erythema, inflammation and modifications of the immune system. These changes have been reported after excessive short-term or long-term exposure to broad spectrum UVB. In this study, we examined the effects of local repetitive UVB irradiation of 311 nm wavelength on the skin of seven young volunteers. Skin biopsies were taken before and after UVB irradiation, and we immunohistochemically analyzed the expression of CD1a and HLA-DR antigens of Langerhans cells (LC), the possible infiltration of dermis/epidermis by CD11b macrophages, the modifications or the induction of intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) involved in the binding of leukocytes to the endothelial surface and the development of perivascular infiltrates of LFA-1⁺ mononuclear cells. We also determined the expression of substance P receptors (SPR) using biotinylated substance P (SPB). Exposure of UVB 311 nm induced a drastic reduction of CD1a⁺ cells and a moderate increase of HLA-DR⁺ dendritic cells in the epidermis without infiltration by CD11b macrophages. An increase of the binding of SPB to upper layer epidermal cells was noted in five of seven biopsies. In the dermis, vessel-associated ICAM-1 expression increased and an induction of E-selectin occurred on nearly 20 to 40% of endothelial cells, but VCAM-1 expression remained undetectable. The percentage of LFA-1⁺ cells did not change significantly after irradiation. These observations may be compatible with a selective role of UVB 311 nm on the skin immune response.     

Acute effects of UVB radiation on the proliferation and differentiation of keratinocytes

PURPOSE: The effects of UVB radiation on the proliferation and differentiation of epidermal keratinocytes were investigated with respect to timing, dosage, and repeated exposures. METHODS: Nine healthy volunteers were placed into three subgroups and exposed to UVB radiation on buttock skin using a Waldmann UV 800 unit fitted with Philips TL-20W/12 fluorescent lamps. Three volunteers were given 2 MED of UVB and biopsied at: pre-exposure, 24, 48 and 72 h after UVB exposure. For three volunteers, 1 MED, 2 MED, 3 MED of UVB were applied. After 48 h, biopsies were taken from non-irradiated and irradiated sites. Finally, three volunteers received 1 MED of UVB daily for 5 days, and the non-irradiated and irradiated sites were biopsied 48 h after the final exposure. The expression of proliferation and differentiation markers by keratinocytes were detected by immunohistochemical staining, and the results were analysed quantitatively by image analysis. RESULTS: The expression of proliferation and differentiation markers was observed prominently 48 h after irradiation. Higher doses of UVB caused an increase in proliferation and differentiation marker expression. Repeated exposures potentiated the effect of UVB radiation. CONCLUSION: UVB irradiation concomitantly promotes epidermal proliferation and differentiation. Responses were maximal 48 h after irradiation. This effect of UVB increases linearly according to dose and repetition.     

细菌衍生黑素对长波紫外线诱导人皮肤成纤维细胞凋亡和坏死的保护

目的:探讨细菌衍生黑素(b-melanin)对长波紫外线(UVA)诱导人成纤维细胞凋亡和坏死产生的光保护作用,为今后将其用作皮肤光保护剂提供依据.方法:正常人成纤维细胞(NL-FB)和着色性干皮病患者成纤维细胞(XP-FB)经UVA照射12 h后,以四甲基偶氮唑蓝(MTT)法检测细胞存活率,Hoechst33258染色法观察早期凋亡细胞核形态学变化.二氯荧光素二酯(DCFH-DA)标记法测定细胞内活性氧基(ROS)水平.结果:UVA照射诱导细胞的半数致死剂量,XP-FB大约为30 J/cm2,而NL-FB＞40 J/cm2.为了观察不同浓度(0,25,50,100,200,400,和800 μg/mL)的b-melanin是否对细胞存在光保护作用,给予半数致死剂量(30 J/cm2)UVA照射后,经100～400 μg/mL b-melanin处理的XP-FB的细胞存活率均较未处理组明显增高(P＜0.01),而NL-FB的细胞存活率变化不明显.细胞内ROS测定结果显示100、400 μg/mL的b-melanin能明显清除UVA诱导产生的ROS.100 μg/mL b-melanin即能阻止非致死剂量(16 J/cm2)UVA照射诱导的早期凋亡细胞核改变.结论:b-melanin能对UVA诱导人成纤维细胞凋亡和坏死提供有效的光保护,这一作用很可能关系到b-melanin对ROS的清除.本研究还首次提出核酸切除修复机制缺陷的XP-FB是一敏感的可用于测试UVA光损伤作用的体外细胞模型.     

Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin by Eisinger and Marko

Melanocytes are pigment producing cells that arise from the neural crest and migrate to the skin early in fetal development. The pigment that melanocytes synthesize, melanin, plays a critical role in protecting the skin from mutagenic ultraviolet irradiation. Melanocytes are also precursor cells for melanoma, a deadly form of skin cancer. Because melanocytes make up a minority population of cells in the epidermis they have been difficult to propagate in culture. The landmark paper by Eisinger and Marko, described below, was the first successful report of large scale propagation of pure cultures of melanocytes. This paper set the stage for an explosive growth in knowledge in the biology of human melanocytes and allowed scientists to begin dissecting the different oncogenic events involved in the transition of melanocytes to melanoma.     

Differential effects of UV irradiation on nuclear retinoid receptor levels in cultured keratinocytes and melanocytes

A major risk factor for skin cancer is UV irradiation, which not only damages DNA and other photosensitive compounds like vitamin A, but may also perturb cellular signaling, e.g. via the retinoid receptor system believed to be important for cancer protection. We used cultured normal human keratinocytes and melanocytes to examine the effects of UV irradiation on the expression of the predominant retinoid receptors in the human skin (RARalpha, RARgamma and RXRalpha) and the AP-1 protein c-Jun; mRNA levels were studied by real-time PCR and protein levels by Western blot. In keratinocytes, a single dose of UVB (50 mJ/cm2) caused a rapid drop in the expression of all three receptors (mRNA levels minus 35-50% after 4 h; protein levels minus 20-45% after 8 h), which was followed over the next 40 h by a variable response, leading to full normalization for RARalpha only. In contrast, the levels of c-Jun did not change significantly after UV exposure. In melanocytes, UVB caused a similar drop of the retinoid receptor levels as in keratinocytes but this was soon followed by an increased expression leading to a complete normalization of all receptor levels within 1-3 days. The c-Jun levels in melanocytes increased 1 day after UV exposure and remained high (plus 50%) thereafter. In both cell types, a approximately 3-fold increase in apoptosis (measured by DNA fragmentation) was observed 8-48 h after UVB irradiation. In conclusion, a depletion of vitamin A and retinoid receptors by UV irradiation, together with unchanged or even increased c-Jun levels, might seriously interfere with retinoid signaling and thus promote future tumor development, especially in keratinocytes.     

内皮缩血管肽1和促肾上腺皮质激素对体外培养的正常人黑素细胞增殖的影响

目的 了解内皮缩血管肽1及促肾上腺皮质激素(ACTH)对体外培养的正常人黑素细胞增殖的影响.方法 采用体外黑素细胞培养技术、四甲基偶氮唑蓝比色法(MTT)研究不同浓度内皮缩血管肽1、ACTH对黑素细胞增殖的影响.结果 从0.1~1000nmol/L浓度的内皮缩血管肽1均可不同程度地促进正常人黑素细胞的增殖,低浓度0.1nmol/L内皮缩血管肽1具有很好的促增殖作用.10^-13~10^-9mol/L的ACTH也可不同程度地促进正常人黑素细胞的增殖,与对照组相比,P值均<0.05,其中又以高浓度10^-9mol/LACTH促增殖能力最强,10^-12mol/L、10^-11mol/L、10^-10mol/L 3个浓度间差异无统计学意义.结论 内皮缩血管肽1及ACTH具有促进体外培养的正常人黑素细胞增殖的作用,较低浓度内皮缩血管肽1即具有很好的促增殖作用,正常人生理水平(10^-12~10^-11mol/L)及更高浓度ACTH均有较强的促增殖作用.     

Evaluation of ex vivo melanogenic response to UVB,UVA, and visible light in facial melasma and unaffected adjacent skin

BackgroundThe independent role of solar radiation in the differential melanogenesis between melasma and adjacent skin is unknown.ObjectivesTo assess the melanogenic responses of skin with facial melasma and of the adjacent skin to UVB, UVA, and visible light, in an ex vivo model.MethodsThis was a quasi-experimental study involving 22 patients with melasma. Facial melasma and adjacent skin samples were collected and stored in DMEM medium, at room temperature. One fragment was placed under the protection from light, while another was exposed to UVB, UVA, and visible light (blue-violet component): 166 mJ/cm², 1.524 J/cm², and 40 J/cm², respectively. Subsequently, all samples were kept for 72 hours in a dark environment and stained by Fontana-Masson to assess basal layer pigmentation, dendrites, and melanin granulation.ResultsEffective melanogenesis was observed in the basal layer in melasma and in the normal adjacent skin after all irradiations (p < 0.01), with the following median increment: UVB (4.7% vs. 8.5%), UVA (9.5% vs. 9.9%), and visible light (6.8% vs. 11.7%), with no significant difference between anatomical sites. An increase in melanin granulation (coarser melanosomes) was observed only after irradiation with UVA and only in the skin with melasma (p = 0.05). An increase in the melanocyte dendrite count induced by UVB radiation was observed in both anatomical sites (p ≤ 0.05).Study limitationsUse of an ex vivo model, with independent irradiation regimes for UVB, UVA, and visible light.ConclusionsMelanogenesis induced by UVB, UVA, and visible light was observed both in melasma and in the adjacent skin. The morphological patterns suggest that different irradiations promote individualized responses on the skin with melasma.     

UVB light and 17-beta-estradiol have different effects on the mRNA expression of Ro/SSA and La/SSB autoantigens in HaCaT cells

Antibodies produced against the Ro/SSA and La/SSB autoantigens are not only of diagnostic value but they may even play a role in the pathogenesis of several autoimmune diseases (Sj?gren's syndrome, subacute cutaneous lupus erythematosus, neonatal lupus erythematosus and systemic lupus erythematosus). Among other factors, ultraviolet (UV) radiation and also the hormonal milieu are well-known cofactors in the pathogenesis of these autoimmune diseases. The goal of our research was to study the possible alterations in mRNA levels of three different Ro antigens and that of two La species produced by alternative splicing in transformed human keratinocytes (HaCaT cells) after UVB irradiation and after 17-beta-estradiol treatment. The polymerase chain reaction technique was used to determine the mRNA levels of the Ro and La species after 24, 48, and 72 h of irradiation. The mRNA levels of calreticulin increased as a function of time after UV irradiation but the mRNA levels of 52 kDa and 60 kDa Ro mRNAs were unaltered. After treating the cells with 17-beta-estradiol, there was no change observed in the levels of Ro mRNAs or La exon 1 mRNA, but a gradual decrease was noted in the mRNA levels of La exon 1'. The importance of alterations in the ratio of La exon 1 to exon 1' is supported by the observations in patients with Sj?gren's syndrome, and our results strengthen the notion that the Ro and La antigens participate in the pathogenesis of different autoimmune diseases.     

Enhanced proliferation and collagen synthesis of human dermal fibroblasts in chronically photodamaged skin

Cutaneous aging can be divided into intrinsic aging and photoaging. We investigated the influence of aging and photoaging on the proliferation and collagen synthesis of human dermal fibroblasts cultured 3-dimensionally in a collagen gel. We examined 11 human dermal fibroblast cell lines cultured from 3 newborn skins (1 day old), and both exposed and unexposed skin from 4 elderly volunteers (60, 60, 73, 76 years old), respectively. Newborn fibroblasts actively proliferated within the attached collagen gels compared with the elderly cell lines. Within the attached collagen gels in the presence of 10% fetal calf serum (FCS), the fibroblasts from exposed skin proliferated rapidly compared with fibroblasts from unexposed skin from the same individuals. In collagen gel and monolayer cultures with 1% FCS, the percentage of collagen synthesized by photoaged and aged fibroblasts decreased significantly compared with that by newborn fibroblasts. When the fibroblasts were cultured three dimensionally in attached collagen gels in the presence of 1% FCS, the relative levels of collagen synthesis by cultured fibroblasts from photoaged skin were increased significantly compared with those of aged skin fibroblasts from the same individuals. These results suggest that fibroblasts of exposed skin may be more active than those of unexposed skin and that the three-dimensional culture of fibroblast can be used as a model to investigate the influence of aging and photoaging on cell functions.     

Differential modulation of stress-inflammation responses by plant polyphenols in cultured normal human keratinocytes and immortalized HaCaT cells

Background

Environmental and endogenous stresses to skin are considered causative reasons for skin cancers, premature ageing, and chronic inflammation. Screening of substances with preventive and/or curative properties is currently based on mechanistic studies of their effects towards stress-induced responses in skin cell cultures.

Objective

We compared effects of plant polyphenols (PPs) on the constitutive, UVA-, LPS-, or TNF-alpha-induced inflammatory responses in cultured normal human epidermal keratinocytes (NHEK) and immortalized HaCaT cells.

Methods

Representatives of three classes of PPs, flavonoids, stilbenoids, and phenylpropanoids were studied. Their effects on mRNA were determined by qRT-PCR; protein expression was assayed by Western blot and bioplexed ELISA; phosphorylation of Akt1, ERK1/2, EGFR, and NFkappaB was quantified by intracellular ELISA or Western blot.

Results

PPs or their combination with UVA or LPS induced strong up-regulation of stress responses in HaCaT but not in NHEK. In addition, compared to NHEK, HaCaT responded to TNF-alpha with higher synthesis of MCP-1, IP-10 and IL-8, concomitant with stronger NFkappaB activation. PPs down-regulated the chemokine release from both cell types, although with distinct effects on NFkappaB, Akt1, ERK, and EGFR activation.

Conclusion

Results of pharmacological screenings obtained by using HaCaT should be cautiously considered while extending them to primary keratinocytes from human epidermis.     

Comparison of broadband UVB, narrowband UVB, broadband UVA and UVA1 on activation of apoptotic pathways in human peripheral blood mononuclear cells

BACKGROUND/PURPOSE: Ultraviolet (UV) radiation is an important therapy for immune-mediated cutaneous diseases. Activation of early apoptotic pathways may play a role in the clinical effectiveness. Different UV wavelengths have different efficacy for various diseases, but it remains unclear whether the ability to induce apoptosis differs with respect to the wavelength, and whether they induce apoptosis through the same mechanism. The aim of this study is to analyze the effects of different UV wavelengths that are used clinically on normal human peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs were treated with UV-light sources broadband UVB, narrowband UVB, broadband UVA and UVA1. Initiation of apoptosis was assessed by flow cytometry by staining-treated cells for activated caspases. Immunoblots were performed to measure for cleaved caspase-3, -8, -9, cytochrome c, Bcl 2-interacting domain and poly-(ADP ribose) polymerase cleavage. RESULTS: We demonstrate that all the UV radiation sources induced caspase activation in a dose-and time-dependent manner. Components of both the extrinsic and intrinsic pathways of apoptosis were activated by all of the UV wavelengths tested, but differed in the level of energy needed for activation. CONCLUSION: The greater effectiveness of UVB on initiation of apoptotic pathway suggests that apoptosis may play a role in the clinical efficacy of UVB-responsive inflammatory cutaneous diseases.     

Age-dependent response of energy metabolism of human skin to UVA exposure: an in vivo study by 31P nuclear magnetic resonance spectroscopy

Purpose The aim of our study was to evaluate the in vivo energy metabolism of human skin as a function of age, in conditions of rest and after a mild stress caused by a suberythemal UVA irradiation. Methods The kinetics of UVA-induced modifications in high-energy phosphorylated metabolites of young and old skins were non-invasively monitored over a period of 24 h using ³¹P nuclear magnetic resonance spectroscopy. In vivo³¹P spectra were obtained on the ventral aspect of the wrist, using a NMR Imaging Spectrometer equipped with a double-tuned surface coil. Concentrations of phosphocreatine, inorganic phospate, adenosine tri-phosphate, phosphomono and phosphodiesters were calculated from the spectra and results were expressed as relative concentrations. A total of 20 subjects were enrolled in this study (n = 10 for the age group below 25 years and n = 10 for the age group above 55 years). A second experiment was then performed on 10 old subjects (mean age 60) who were treated on one wrist, twice a day for one month prior to UVA irradiation, with a product that contained active ingredients to restore barrier function and modulate the inflammatory response, the other wrist being an untreated control. Results Baseline levels of phosphorylated metabolites were similar in young and old skins. A suberythemal dose of UVA (6 J.cm⁻²) led to a significant decrease in the PCr/Pi ratio (index of energy status) and a significant increase in the PME/PDE ratio (index of cellular turnover rate of lipid-related metabolites) within 1 h. The observed variations were transient and the recovery was complete at T + 24 h post-UVA, although recovery was significantly slower in the older group. The disturbances were significantly reduced after treatment of the older skin with a formula that restored barrier function of the stratum corneum and modulated the inflammatory response. Conclusion (i) baseline levels of energy metabolites in skin do not seem to vary with age; (ii) low dose UVA irradiation induces a rapid response in the energy metabolism of the skin; (iii) the kinetics of the response and recovery after an aggression by UVA suggest that older skin has significantly less energy rebound after a stress situation than younger skin; (iv) the energy reserve in older skin can be protected efficiently against UVA-induced stress by restoring barrier function and modulating the inflammatory response.