Effect of narrow-band ultraviolet B radiation on the autophagy of cultured human melanocytes in vitro / 中华皮肤科杂志

Objective To evaluate the effect of narrow-band ultraviolet (NB-UVB) radiation on the autophagy of cultured human melanocytes in vitro,and to explore possible mechanisms underlying the treatment of vitiligo by NB-UVB.Methods In vitro cultured human melanocytes were divided into 4 groups to be irradiated with NB-UVB at different irradiation doses of 0 (control group),50,100 and 200 mJ/cm2 (50-,100-and 200-mJ/cm2 NB-UVB groups) respectively.After 24-hour treatment,the cells were collected,and monodansylcadaverin (MDC) staining was conducted to detect changes of autophagosomes in melanocytes.Western blot analysis was performed to determine the protein expression of autophagy signals including phosphorylated AMP-activated protein kinase (p-AMPK),phosphorylated mammalian target of rapamycin (p-mTOR),microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ (LC3 Ⅱ/Ⅰ) and P62,and transmission electron microscopy to observe ultrastructural changes of autophagosomes and melanosomes in the melanocytes.Statistical analysis was done by using one-way analysis of variance (ANOVA) for the comparison of Western blot results,and by Kruskal-Wallis H test for the comparison of the number of melanosomes,autophagosomes and autolysosomes.Results MDC staining showed that the percentages of autophagosome-positive melanocytes were significantly higher in the 100-,200-mJ/cm2 NB-UVB groups (38.08％ ± 4.10％,40.23％ ± 1.45％,respectively) than in the control group (21.83％ ± 3.50％,both P ＜ 0.05) and 50 mJ/cm2 NB-UVB group (23.66％ ± 4.12％,both P ＜ 0.05).As Western blot analysis revealed,the 100-,200-mJ/cm2 NB-UVB groups showed significantly increased expression of p-AMPK and LC3 Ⅱ/Ⅰ,but significantly decreased expression of p-mTOR and P62 compared with the control group (all P ＜ 0.05).Transmission electron microscopy showed that the number of autophagosomes and autolysosomes was significantly higher in the 100-,200-mJ/cm2 NB-UVB groups (5.12 ± 1.13,5.25 ± 1.04) than in the control group (1.88 ± 1.18,both P ＜ 0.05).Meanwhile,the number of melanosomes was significantly higher in the 50-,100-and 200-mJ/cm2 NB-UVB groups (39.12 ± 9.42,57.38 ± 7.11,59.75 ± 15.15,all P ＜ 0.05) than in the control group (18.50 ± 4.18,all P ＜ 0.05).Conclusion NB-UVB radiation can not only promote the formation of melanosomes,but also activate the autophagy signal pathways in the melanocytes and promote the formation of autophagosomes and autolysosomes,which may be one of the mechanisms underlying the treatment of vitiligo by NB-UVB.

Expression and activity of cathepsin L2 and its effect on the degradation of melanosomes in skin lesions of seborrheic keratosis / 中华皮肤科杂志

Objective To determine the expression of cathepsin L2 (CTSL2)and evaluate its activity in skin lesions of seborrheic keratosis (SK),to observe the ultrastructural changes of melanosomes in the skin lesions of SK,and to estimate the effect of CTSL2 on the degradation of melanosomes.Methods Twenty patients with SK were enrolled from the Department of Dermatology,Renmin Hospital of Wuhan University.The lesional tissue and the perilesional normal skin were biopsied from each patient.Among 15 of the 20 patients,hematoxylin and eosin (HE)staining and Fontana-Masson silver staining were performed to observe the distribution of melanin granules,transmission electron microscopy (TEM)was conducted to observe the ultrastructural changes of melanosomes,and immunohistochemical staining was performed to estimate the cellular proliferative activity.RT-PCR and fluorogenic substrate cleavage assay were performed in the other 5 patients to determine the mRNA expression of CTSL2 and evaluate its activity,respectively.Sucrose density gradient ultracentrifugation was performed to isolate and purify melanosomes from the retinal pigment epithelium (RPE) harvested from a discarded eyeball of a 35-year old male patient with informed consent.The purified melanosomes were incubated with epidermal lysates of SK lesions,and TEM was used to observe the changes in the membrane structure of melanosomes.Statistical analysis was carried out by paired t test,and a P value ＜ 0.05 was considered statistically significant.Results A large number of melanin granules were deposited in SK lesions,while the linear deposition of melanin granules was only seen in the basal layer of the normal skin.TEM showed that the percentage of damaged melanosomes was much higher in the normal skin (49.00％ ± 4.00％) than in the SK lesions (24.33％ ± 3.06％)(t =8.49,P ＜ 0.05).RT-PCR revealed that the mRNA expression and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin (mRNA:0.35 ± 0.09 vs.0.43 ± 0.08,t =3.17,P ＜ 0.05;activity:17.46 ± 0.45 vs.28.78 ± 0.58,t =34.29,P ＜ 0.05).Moreover,TEM also showed that the percentage of damaged melanosome was lower in the SK lesion lysate-treated group (32.33％ ± 4.93％) than in the normal skin lysate-treated group (43.00％ ± 2.65％,t =3.30,P ＜ 0.05).Conclusion Decreased expression of CTSL2 in the SK lesions can affect the degradation of melanosomes by keratinocytes.However,whether CTSL2 directly takes part in the pathogenesis of SK or not is still needed to be further confirmed.

Lab color measurements of Ota nevus, freckles and melasma / 中华医学美学美容杂志

Objective Lab color mode can be used to digitize the color.This study explores the possibility of using Lab model to measure facial pigmentations.Methods Lab color model was used to measure the normal skin and three common clinical pigmentations (Ota nevus,freckles and melasma).We also analyzed the characteristics and assessed the data changes after the treatment.Results Average L,a and b values were 54.4,13.8 and 19.0 in normal skin,34.6,5.17 and 6.9 in Ota nevus,43.25,16.15 and 23.05 in freckles and 40.5,16.8 and 23.35 in melasma,respectively.The Lab values of freckles and melasma were close.The order of L value was:normal skin ＞ freckle ＞ melasma ＞Ota nevus;the order of value of a and b was:melasma ＞ freckle ＞ normal skin ＞ Ota nevus.After treatment,the Lab values gradually tended to be the values of normal skin.Conclusions The Lab color mode can be used as a digital description method for skin color and facial pigmentation,which provides an objective measure for clinical research.

Noncompetitive antagonists and agonists of glutamate receptor regulate filopodia formation in epidermal cells and melanosome transfer: an experimental study / 中华皮肤科杂志

Objective To investigate the regulatory effect of glutamate signaling pathway on filopodia formation in epidermal cells and on melanosome transfer.Methods Epidermal melanocytes and keratinocytes were isolated from human foreskin and subjected to subculture.After two to three passages of subculture,the melanocytes and keratinocytes were cultured alone or in combination with or without the presence of MK801 (an antagonist of N-methyl-D-aspartic acid (NMDA) receptor) of 100 μmol/L,or NMDA (the activator of NMDA receptor) of 100 μmol/L,for 24 hours.The melanocytes irradiated with UVB at 311 nm served as the control.Scanning electron microscopy was used to observe the appearance of filopodia and dendrites of melanocytes and keratinocytes.Melanosome transfer was visualized under confocal laser scanning microscopy after double immunofluorescent staining.Results Although no obvious changes were observed in the number of dendrites in monocultured melanocytes after treatment with MK801 or NMDA for 24 hours,dendrites became thinner at the terminus and longer with a decrease in the number and length of filopodia after MK801 treatment,but thicker and shorter with an increase in the number and length of filopodia after NMDA treatment compared with untreated monocultured melanocytes.In the coculture system,filopodia were observed between the untreated melanocytes and keratinocytes,and the number of filopodia in melanocytes was larger in the side adjacent to keratinocytes than in the opposite side.Compared with the untreated coculture system,the number of both filopodia connecting melanocytes and keratinocytes and filopodia extending from melanocytes to keratinocytes decreased in the coculture system after treatment with MK801 of 100 μmol/L,but increased after treatment with NMDA of 100 μmol/L,for 24 hours.Melanosomes were found in keratinocytes cocultured with melanocytes without treatment,which were decreased in number after 24-hour treatment with MK801 of 100 μmol/L,but increased in number and even present in keratinocytes nonadiacent to melanocytes after 24-hour treatment with NMDA of 100 μmol/L.Conclusion Glutamate signaling pathway may modulate the transfer of melanosomes from melanocytes to keratinocytes via modulating the appearance of melanocyte dendrites and formation of filopodia.

Classificação clínica da pele: avaliação crítica / Skin clinical classification: critical analysis

Os autores propõem a análise da classificação clínica da pele, considerando a evolução do homem dentro de diferentes locais geográficos no planeta. Estudos histológicos sugerem ser a origem do homem única, tendo como base a quantidade de melanócitos, que é semelhante nos diferentes tipos de pele, variando apenas nas características dos melanossomos. Verifica-se que a diferença entre a melanina da epiderme de um caucasoide e de um negroide se encontra, de fato, nas características dos melanossomos. Relatam, também, as várias tentativas de classificar os tipos de pele, mostrando que até hoje não existe uma classificação plenamente satisfatória.

Ultrastructure and transcriptional activity of MITF in epidermal melanocytes from patients with vitiligo / 中华皮肤科杂志

Objective To analyze the relationship of melanocyte ultrastructure and expression of microphthalmia-associated transcription factor (MITF) as well as tyrosinase-related proteins (TRP) transcriptionally modulated by MITF to clinical types and duration of vitiligo.Methods Epidermal sheets were taken by suction blisters respectively from lesional,perilesional,and normal skin of 12 patients with vitiligo vulgaris (VV) and 8 with segmental vitiligo (SV).The duration of vitiligo varied from 3 to 300 months in these patients. Transmission electron microscopy was performed in 10 patients with vitiligo,including 6 cases of VV and 4 cases of SV.Epidermal melanocytes from normal skin of 20 patients were subjected to culture followed by Western blot to detect the expression level of MITF and some molecules transcriptionally modulated by MITF,including tyrosinase (TYR),TYR-related protein-1(TYRP1),and TYR-related protein-2 (TYRP2) in cultured melanocytes.Results Epidermal melanocytes were absent in lesional skin of 7 out of 10 patients observed for ultrastructural alterations,whereas melanocytes with reduced or absent melanin (melanosome) could accidently be seen in lesional skin of 1 patient with short-standing vitiligo and 2 patients with long-standing vitiligo.In perilesional skin.abnormal ultrastructure of melanocytes was found in 3 with a duration of vitiligo less than 15 months among 6 patients with VV,and in 1 out of 4 patients with SV.The down-regulated expression of MITF was consistent with that of TYR,TYRP1 and TYRP2 in cultured epidermal melanocytes from normal skin of patients with VV;in those from patients with SV,the down-regulated expression was observed only in MITF,while the expressions of TYR,TYRP1 and TYRP2 were nearly normal.Conclusion Differences may exist between VV and SV in the ultrastructure as well as mechanisms of transcriptional modulation by MITF in epidermal melanocytes.

Assessment of melanosome transfer by selective incorporation of 14C-thiouracil into nascent melanin / 中华皮肤科杂志

Objective To establish a method to quantitatively assess melanosome transfer with incorporation of 14C-thiouracil (TU) into nascent melanin. Methods To characterize whether 14C-TU was exclusively incorporated into melanin-producing cells, the same number of mouse melan-a or SP-1 keratinocytes were labeled with 14C-TU for 12 hours and 48 hours, respectively, followed by the measurement of radioactivity. Mouse melan-a melanocytes were pre-labeled with 1 Ci/mL 14C-TU, and cocultured with mouse SP1 keratinocytes to develop an assay system for melanosome transfer to keratinocytes. Following co-culture, the keratinocytes with transferred radioactivity were separated from melanocytes at different time points via two times of differential trypsinization. Transferred radioactivity in keratinocytes, denoting the amount of melanosome transfer, was measured with liquid scintillation counting. Meanwhile, the effects of forskolin, a PKA activator, and nicotinamide on melanosome transfer were also investigated with this assay system.Results The incorporated radioactivity in melan-a cells was 66- or 80-fold as high as that in SP-1 cells,indicating that 14C-TU would be a suitable tracer for melanosome transfer in co-culture with keratinocytes. A purity of 84.5% was achieved for keratinocytes with transferred radioactivity by twice differential trypsinization.As shown by this assay, there was an approximately 0.67-fold decrease in melanosome transfer with the treatment of 1 g/L nicotinamide and 2.3-fold increase with 20μmol/L forskolin treatment. After coculture with SP1 cells for 8-12 hours, melan-a cells developed well-extending dendrites with detectable melanosome transfer, while no proliferation of melan-a cells induced by forskolin was seen. Conclusion An optimized protocol for selective incorporation of 14C-TU into nascent melanin has been successfully applied to the quantitative measurement of melanosome transfer from melanocytes to keratinocytes induced by forskolin or nicotinamide.

Estudo comparativo morfofuncional de melanócitos em lesões de melasma / Morphological and functional comparative study of melanocytes in melasma lesions

FUNDAMENTOS - Melasma é hipermelanose comum caracterizada por máculas acastanhadas em áreas fotoexpostas, cuja fisiopatogenia não é totalmente esclarecida. OBJETIVOS - Caracterizar e comparar morfologica e funcionalmente os melanócitos da epiderme sã com os da pele afetada por melasma. MÉTODOS - Avaliaram-se 12 pacientes portadores de melasma facial, sendo realizadas biópsias da pele lesada e pele sã adjacente. Os cortes foram corados por hematoxilina-eosina, Fontana-Masson, marcados pelo Melan-A e submetidos à microscopia eletrônica. A quantificação epidérmica de melanina e melanócitos foi estimada a partir de análise citomorfométrica digital. RESULTADOS - Todas as pacientes eram mulheres com média de idade 41,3±2,8 anos. Ao Fontana-Masson evidenciou-se importante aumento da melanina epidérmica na pele lesada em relação à pele sã. A marcação pelo Melan-A demonstrou melanócitos maiores com dendritos proeminentes na pele lesada. Observou-se maior densidade de melanina epidérmica na pele lesada, e a análise digital do número de melanócitos da epiderme não demonstrou diferença significativa entre pele lesada e sã. À microscopia eletrônica, observaram-se número aumentado de melanossomas maduros nos ceratinócitos e melanócitos com organelas citoplasmáticas proeminentes na pele lesada. CONCLUSÕES - Melanogênese aumentada na epiderme com melasma em relação à epiderme normal adjacente.

BACKGROUND - Melasma is a common hypermelanosis characterized by symmetric brownish macules on photoexposed areas, most frequently on the face of women. Its pathophysiology is still unknown. OBJECTIVES - To morphologically and functionally characterize and compare melanocytes of normal skin and of melasma. METHODS - Twelve patients with facial melasma were assessed and biopsies of lesions and adjacent healthy skin were performed. The slices were stained with hematoxylin-eosin and Fontana-Masson, immunohistochemically marked for Melan-A and evaluated by electronic microscopy. Quantification of epidermal melanin and melanocytes was estimated by digital cytomorphometric analysis. RESULTS - All patients were female, mean age of 41.3±2.8 years. The Fontana-Masson staining showed an important increase in epidermal melanin as compared to normal skin. The Melan-A staining demonstrated larger and intensely marked melanocytes and more prominent dendrites in the damaged skin. Greater epidermal melanin density was observed in melasma lesions and the digital analysis of number of epidermal melanocytes did not show a significant difference between damaged and normal skin. Electronic microscopy analysis revealed an increased number of mature melanosomes in keratinocytes and melanocytes, with marked cytoplasmic organelles in melasma skin. CONCLUSIONS - Melanogenesis is increased on melasma epidermis as compared to adjacent normal skin.

Ultrastructural Characteristics of Amelanotic Melanocytes from Human Hair Follicles / 中华皮肤科杂志

Objective To investigate the ultrastructural characteristics of amelanotic melanocytes (AMMCs). Methods Individual hair follicles from normal human scalp were digested with collagenase type V, then washed in phosphate buffer saline. Hair-follicle cell suspensions were prepared by trypsin and cultured in a medium suitable for melanocyte growth. The keratinocytes were removed by differential trypsinization. Geneticin (100?g/mL) was used to eliminate contaminating fibroblasts. After 3 passages the cells were trypsinized, washed in phosphate buffer saline, and finally processed for transmission electron microscopy. Results Under transmission electron microscope, the cultured cells were round or oval-shaped with a single large nucleus and double-layered karyotheca. Abundant euchromosome but sparse heterochromosome was observed within the nucleus. There were various organelles in the cytoplasm, including mitochondria, rough endoplasmic reticulum (RER), ribosomes and abundant melanosomes of nearly uniform size. The electronic density granules distributed in a concentric pattern in most of the melanosomes. Colgi complexes were inconspicuous in the cells. Conclusions Compared to epidermal melanocytes, AMMCs from human hair follicles have different ultrastructural characteristics which implies their functional immaturity. AMMCs may serve as the depot for mature melanocytes.

The ultraviolet B protection effects of topically applied melanosomes onto human skin

Melanosome is a cellular organelle that is composed of a melanosomal matrix and a brown biochrome, melanin which is formed by tyrosine-tyrosinase reactions. The melanosome is formed within the melanocyte and transferred to the surrounding keratinocytes through dendritic processes. Human skin color is related to the number, size, type and distribution of melanosomes, and the major role of melanosomes is to prevent skin from injurious nonionizing ultraviolet radiation. Controlled NaOH hydrolysis and centrifugation of human hair make it possible to isolate large amounts of melanosomes which are synthesized within the follicular melanocytes and transferred to hair matrix cells. In this study, the sun protection factors of topically applied melanosomes isolated from human hair were evaluated using ultraviolet B phototesting. Topically applied melanosomes increased the minimal erythemal doses. And the sun protection factors of each 50% and 25% melanosomal preparation were 12.3 +/- 5.5 and 3.1 +/- 1.3 respectively, and these ultraviolet B protection effects showed statistically significant differences from 10%, 5% and 1% melanosomal preparations and vehicle. Form these results, the dose-related photoprotective role of melanosomes was confirmed.

Isolation and Identification of Melanosomes from Human Hair

Melanosomes were isolated from the human hair by graded centrifugation and identified by transmission and scanning electron microscopic examination. Melanosomes were separated from the keratinous structures by treating with strong NaOH solution for 15 hours. The keratinous structures were removed by centrifugation ai 2,500xg and 3,500xg for 10 minutes respectively at 0 ℃. The isolated melanosomes were collected by centrifugation at 7,800xg at 0 ℃. Scanning electron microscopic examination made it possible to evaluate the global structure of purified melanosomes.