Objective:To assess the value of culture of epidermal melanocytes from negative-pressure suction blisters in the auxiliary diagnosis of segmental vitiligo-like nevus depigmentosus.Methods:Between June 2019 and March 2020, 8 patients with segmental vitiligo-like nevus depigmentosus, who met the Coupe′s clinical diagnostic criteria, were enrolled from Department of Dermatology, Hangzhou Third People′s Hospital. All patients were evaluated by the Wood′s lamp, reflectance confocal microscopy (RCM) , 308-nm excimer laser radiation, and in vitro culture of epidermal melanocytes from negative-pressure suction blisters. Results:Among the 8 patients, fluorescence was observed in 6 under the Wood′s lamp, dermal papillary rings were incomplete or absent in 4 as shown by RCM, and 5 experienced no repigmentation after 308-nm excimer laser radiation. Among the 8 patients, in vitro cultured lesional melanocytes were all positive for ferrous sulfate staining, yellowish-white precipitates were obtained after digestion and centrifugation of the melanocytes, and stage Ⅰ-Ⅲ melanosomes were observed in the cytoplasm of melanocytes under the electron microscope; however, the precipitates were black in color after digestion and centrifugation of the melanocytes collected from the normal skin tissues at the contralateral anatomical site, and stageⅠ-Ⅳ melanosomes were seen in the cytoplasm of the melanocytes under the electron microscope. Conclusion:Culture of epidermal melanocytes from negative-pressure suction blisters may facilitate the diagnosis of segmental vitiligo-like nevus depigmentosus.

Objective:To investigate clinical efficacy and safety of cultured autologous melanocyte transplantation for the treatment of non-segmental vitiligo accompanied by autoimmune thyroid diseases.Methods:From May 2008 to December 2018, a total of 2 284 patients with non-segmental vitiligo were retrospectively collected, who received cultured autologous melanocyte transplantation in Hangzhou Third People′s Hospital. Among these patients, 75 were also diagnosed with autoimmune thyroid diseases, including hyperthyroidism (42 cases) , hypothyroidism (18 cases) and Hashimoto′s thyroiditis (15 cases) . Efficacy and safety were compared between the vitiligo patients with autoimmune thyroid diseases (concomitant group) and those without (non-concomitant group) . Chi-square test was used to compare enumeration data.Results:Among the 2 284 patients, 1 085 were males and 1 199 were females, with an age of 25.0 ± 1.2 years and a disease duration of 5.1 ± 2.3 years. Six months after transplantation, 1 873 out of 2 209 patients in the non-concomitant group achieved favorable clinical response, with a response rate of 84.8%, including 1 162 achieving complete clinical response (52.6%) ; 46 out of 75 patients in the concomitant group achieved favorable clinical response, with a response rate of 61.3%, including 20 achieving complete clinical response (26.7%) ; the response rate and recovery rate were both significantly lower in the concomitant group than in the non-concomitant group ( χ2 = 29.72, 19.54, respectively, both P < 0.001) . Moreover, the response rate was significantly lower in the hypothyroidism group than in the hyperthyroidism group ( χ2 = 6.61, P = 0.010) . The incidence of isomorphic response at the donor site was significantly higher in the concomitant group than in the non-concomitant group (9.3% vs. 4.3%, χ2 = 4.31, P = 0.038) , so were the recurrence rates of vitiliginous patches at the recipient site after 1, 3, 5 and 10 years (concomitant group: 6.7%, 14.7%, 17.3%, 8.7%, respectively; non-concomitant group: 0.7%, 1.4%, 2.1%, 3.6%, respectively; χ2 = 29.96, 70.69, 67.23, 41.61, respectively, all P < 0.001) . Conclusion:Concomitant autoimmune thyroid diseases negatively affect the efficacy of cultured autologous melanocyte transplantation in the treatment of vitiligo, so effective measures should be taken to prevent isomorphic response and recurrence at the recipient site for patients with non-segmental vitiligo complicated by autoimmune thyroid diseases.

Objective:To study the characteristics of intestinal microflora in patients with vitiligo, and to analyze the relationship between the changes of intestinal microflora and the incidence of vitiligo, so as to provide new ideas for clinical treatment.Methods:Fecal specimens from 30 patients with vitiligo and 30 healthy adults were collected and analyzed qualitatively by Roche/45 high-throughput sequencing platform. At the same time, macrogenomics was used to analyze the feces of 5 patients with vitiligo and 5 healthy adults to identify the potential regulatory pathways.Results:The bacterial species in the feces of patients with vitiligo were similar to those of healthy people, but the intestinal microbial diversity of patients with vitiligo was significantly reduced ( P<0.01); the abundance of Proteus and Clostridium was significantly reduced at phylum level; at genus level, 7 of them were Bacteroides, Escherichia coli Shigella, Rochella, Bacillus anthracis, Clostridium clostridium, Jordani bacteria. The abundance of RF9 and Prunella-7 decreased significantly ( P<0.01), while the abundance of 4 genera (Rumen Coccus-1, Rumen Coccus UCG, Trichomonas and Streptococcus) increased significantly ( P<0.01). The expression of Streptococcus and Phase Anthraceae in vitiligo patients was significantly different: the former increased by 10.8 times, the latter decreased by 6.517 times, and an intestinal microorganism based on 11 vitiligo-related genera was constructed. The random forest model of bacterial flora showed that AUC of the discriminant model was 0.89 in ROC, and macrogenomic analysis showed that the disorders of vitiligo-related bacterial flora were mainly related to immune-related pathways (such as WNT pathway, Notch pathway), energy metabolism, mitochondrial function and amino acid metabolism (such as phenylalanine metabolism). Conclusions:The diversity of bacterial community in intestinal microecological environment of vitiligo patients is significantly different from those in normal people. The imbalance of bacterial community may be involved in the pathogenesis and development of vitiligo. Supplementation of probiotics may be beneficial to the treatment of vitiligo.

Objective:To identify and differentiate cell subsets in the epidermis and dermis of vitiligo skin lesions using single-cell RNA sequencing technology, and to study the relationship between them.Methods:Skin samples were collected from 2 healthy people without immune or systemic diseases and 2 patients with stable non-segmental vitiligo in Department of Dermatology in the Third People′s Hospital of Hangzhou in September 2019. Single-cell transcriptome sequencing was performed on 11 000 cells in all the skin samples by using 10 × Genomics single-cell RNA-Seq technology. Cell subsets were analyzed, screened and counted by using Seurat software.Results:Cluster analysis of gene expression in the 2 normal skin tissues revealed several cell subsets, including keratinocytes, fibroblasts, nerve cells and melanocytes, endothelial cells, tissue stem cells, and immune cells mainly consisting of dendritic cells and T cells. In the 2 vitiligo lesions, abnormal differentiation and quantity were observed in fibroblasts and 4 keratinocyte subpopulations. The proportion of fibroblasts was significantly lower in vitiligo lesions than in normal skin tissues (0 vs. 0.4%) , while the proportions of keratinocyte subpopulations 5, 6, 10 and 12 (8.03%, 7.36%, 3.52%, 0.91%, respectively) in vitiligo lesions were significantly higher than those in the normal skin tissues (4.47%, 3.53%, 2.69%, 0.28%, respectively, all P < 0.01) . Moreover, the above keratinocyte subpopulations were at the end of cell differentiation, and expressed very significant and specific marker genes, which were mainly closely related to cell-cell interactions and cell homeostasis. GO and KEGG analysis showed that keratinocyte subpopulations 5 and 6 were mainly related to intercellular connection, cell adhesion and cytoskeleton function, while the keratinocyte subpopulation 10 was closely related to cell homeostasis. Conclusion:The single-cell sequencing technology was firstly used to study the transcriptional expression profile of vitiligo lesions in China, and preliminary analysis revealed 4 groups of keratinocytes with different quantity and functions, suggesting that abnormal differentiation and dysfunction of keratinocyte subpopulations may affect the occurrence and development of vitiligo.

Objective To evaluate the efficacy of autologous cultured melanocyte transplantation in the treatment of genital vitiligo.Methods A retrospective analysis was carried out.From 2013 to 2016,6 patients with genital vitiligo were enrolled from Department of Dermatology,Hangzhou Third People's Hospital,who received the treatment with autologous cultured melanocyte transplantation.These patients aged 13-29 years,and the age of onset and disease duration ranged from 10 to 19 years and 2 to 10 years respectively.The area of white patches at the genital site ranged from 10 to 25 cm2.Before the transplantation,all the white patches of the 6 patients bad been stable for ＞ 12 months.Two weeks after the treatment,pimecrolimus was applied to the wounds twice a day after crust shedding.When the patient achieved ＞ 90％ repigmentation of the white patches,pimecrolimus was applied once per three days for maintenance treatment.After the treatment,these patients were followed up for at least 24 months,and the longest follow-up period was 60 months.Results Obvious pigmentation was observed in all the 6 patients 2 months after the transplantation.Meanwhile,3 patients achieved 80％ repigmentation,2 achieved 70％ repigmentation,and 1 achieved 50％ repigmentation.Six months after the treatment,3 patients achieved 95％ repigmentation,2 achieved 80％ repigmentation,and 1 still maintained 50％ repigmentation.Twelve months after the treatment,95％ repigmentation was still maintained in the 3 patients,90％ repigmentation was achieved in 2 patients,and 1 patient maintained 50％ repigmentation,and these outcomes lasted until the end of follow-up.During the follow-up,no adverse reactions were observed.Conclusion Autologous cultured melanocyte transplantation is effective for the treatment of stable genital vitiligo.

Objective To investigate the value of the severity of isomorphic response at the donor site in evaluation of the therapeutic effect of cultured melanocyte transplantation.Methods A total of 172 vitiligo patients,who received cultured melanocyte transplantation or non-cultured epidermal suspension transplantation at the Department of Dermatology of Hangzhou Third Hospital from May 2008 to August 2016 and developed isomorphic response at the donor site,were enrolled into this study.According to the area of isomorphic response,these patients were divided into 2 groups:incomplete isomorphic response group whose area of isomorphic response was less than the suction area,and complete isomorphic response group whose area of isomorphic response was equal to the suction area.The correlation between therapeutic effects of melanocyte transplantation and isomorphic response was analyzed.Results Of the 172 patients,83 had incomplete isomorphic response,and 89 had complete isomorphic response at the donor site.In the incomplete isomorphic response group,21 (25.3％) patients were cured,and 17 (20.5％) were markedly improved,while 4 (4.5％) patients were cured,and 11 (12.4％) were improved in the complete isomorphic response group.Additionally,the response rate was significantly higher in the incomplete isomorphic response group than in the complete isomorphic response group (45.8％ vs.16.9％,x2 =31.581,P ＜ 0.001).Furthermore,the duration of stable phase was also significantly longer in the incomplete isomorphic response group than in the complete isomorphic response group (18.5 ± 15.3 months vs.10.2 ± 7.3 months,t =4.581,P ＜ 0.001).Correlation analysis showed that the duration of stable phase,which was classified into 5 grades including 6-11 months,12-23 months,24-35 months,36-47 months and ≥ 48 months,was negatively correlated with the severity (incomplete or complete) of isomorphic response (rs =-0.322,P ＜ 0.001),but positively correlated with repigmentation rates of the skin lesions (rs =0.675,P ＜ 0.001).Conclusion The length of duration of stable phase is an important factor affecting the therapeutic effect of melanocyte transplantation in vitiligo patients with isomorphic response at the donor site,and the severity of isomorphic response can indicate the length of duration of stable phase and predict the therapeutic effect of melanocyte transplantation.

Objective To investigate the protective effect of acetylated epigallocatechin gallate (AcEGCG) against H2O2-induced oxidative damage to human epidermal melanocytes,and to explore its possible mechanism.Methods Human epidermal melanocytes were isolated and cultured in vitro.Some melanocytes were classified into a H2O2 group induced by H2O2 only,EGCG groups and AcEGCG groups induced by H2O2 after pretreatment with different concentrations of EGCG and AcEGCG,respectively.Three concentrations (10,20 and 40 μmol/L) of EGCG or AcEGCG were used to treat melanocytes for 1 hour in MTS assay and lactate dehydrogenase (LDH) leakage assay and for 2 hours in Western blot assay,while only one concentration (40 μmol/L) was used to treat melanocytes for 0.5,1,2 and 4 hours respectively in flow cytometry assay.Some melanocytes treated with only culture medium and 0.1％ dimethyl sulphoxide (DMSO) served as the control group.After additional culture,MTS assay was performed to determine cell survival rate,flow cytometry to detect the level of reactive oxygen species (ROS) in melanocytes,Western blot to measure the expressions of caspase-9 and caspase-3 proteins.Lactate dehydrogenase (LDH) kit was used to detect the leakage of LDH to culture medium.Statistical analysis was carried out by using one-way analysis of variance for comparisons of multiple group means followed by Student-Newman-Keuls-q (SNK-q) test for multiple comparisons.Results Compared with the control group,the H2O2 group showed significantly decreased cell survival rate (22.99％ ± 0.53％,P ＜ 0.01),but increased LDH leakage level (36.58％ ± 0.73％,P ＜ 0.01),intracellular ROS level (19.08 ± 0.57,P ＜ 0.01),as well as caspase-9 (2.65 ± 0.079,P ＜ 0.01) and caspase-3 (2.36 ± 0.057,P ＜ 0.01) expressions.In comparison with the H2O2 group,the cell survival rate was significantly higher in the 10-,20-and 40-μmol/L AcEGCG groups (79.50％ ± 3.62％,86.52％ ± 5.13％,97.81％ ± 5.21％,respectively,all P＜ 0.01) and EGCG groups (43.19％ ± 1.68％,63.34％ ± 3.60％,70.82％ ± 2.1％,respectively,all P ＜ 0.01).However,the 10-,20-and 40-μ mol/L AcEGCG groups and EGCG groups all showed a significant decrease in the expression levels of caspase-9 (AcEGCG groups:1.44 ± 0.067,1.26 ± 0.059 and 1.10 ± 0.072 respectively;EGCG groups:2.31 ± 0.085,2.13 ± 0.091 and 1.35 ± 0.064 respectively,all P ＜ 0.05) and caspase-3 (AcEGCG groups:1.70 ± 0.053,1.57 ± 0.057 and 1.24 t 0.068 respectively,all P＜ 0.05;EGCG groups:2.09 ± 0.076,1.98 ± 0.093 and 1.79 ± 0.056 respectively,all P ＜ 0.05) compared with the H2O2 group.Similarly,a significant reduction was observed in the leakage level of LDH in these AcEGCG and EGCG groups (all P ＜ 0.01) and in ROS levels in the 40-μmol/L AcEGCG and EGCG groups when compared with the H2O2 group.Conclusions AcEGCG has a stronger protective effect against H2O2-induced oxidative damage to human epidermal melanocytes compared with EGCG,which may be realized through clearance of free radicals,antioxidant effects,and decrease of caspase-9 and caspase-3 expressions.

Objective To evaluate the therapeutic efficacy of autologous melanocyte transplantation for the treatment of vitiligo in patients with abnormal thyroid function.Methods A total of 60 patients with vitiligo were enrolled in this study,including 30 with abnormal thyroid function and 30 without.Epidermal sheets were obtained by suction blister biopsy from the normal skin of all the patients followed by melanocyte isolation and culture.After 2-5 passages of subculture,the melanocytes were transplanted onto vitiliginous lesions,which were abraded previously by ultra-pulsed CO2 laser,in the corresponding patients.All the patients were followed for 6-12 months.Results Of the 30 patients with abnormal thyroid function,7 patients achieved more than 90％ repigmentation,9 patients 50％-89％ repigmentation,53.3％ more than 50％ repigmentation,with the average repigmentation rate being 47％ within 6 months after the transplantation.Meanwhile,13 out of the 30 patients without abnormal thyroid function showed more than 90％ repigmentation,11 showed 50％-89％ repigmentation,with the average repigmentation rate being 75％.Both the cure rate and response rate were significantly higher in the patients without abnormal thyroid function than in those with (cure rate,43.3％ vs.23.3％,P＜ 0.05; response rate,80％ vs.53.3％,P＜ 0.05).Significant differences were also found in the response rate for lesions on the face or neck and for those sized more than 20 cm2 between the two groups of patients (both P ＜ 0.05).The lesions transplanted with epidermal melanocytes from the waist exhibited the lowest cure rate and response rate.Conclusion Clinical or subclinical thyroid dysfunction may have a negative impact on the efficacy of autologous melanocyte transplantation in vitiligo.

Objective To investigate the relationship between the efficacy of autologous cultured melanocyte transplantation and serum levels of interleukin-17 (IL-17) and FoxP3 in patients with vitiligo.Methods Forty patients with stable vitiligo vulgaris were included in this study,and received autologous cultured melanocyte transplantation.Six months after the transplantation,treatment efficacy was evaluated,and patients were classified into the successfully treated group (n =25) and unsuccessfully treated group (n =15).Peripheral blood was collected from all the patients before the transplantation,and enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum levels of IL-17 and FoxP3.Statistical analysis was done using two independent samples t-test with the SPSS software (version 17.0).Results The successfully treated patients showed lower serum levels of IL-17 ((15.29 ± 7.86) vs.(43.88 ± 13.02) ng/L,P ＜ 0.05),but higher serum levels of FoxP3 ((6.08 ± 2.03) vs.(3.37 ± 1.81) ng/L,P ＜ 0.05) than the unsuccessfully treated patients.Conclusion The increased serum IL-17 and decreased serum FoxP3 may contribute to the failure of autologous cultured melanocyte transplantation in patients with vitiligo.

Objective To evaluate the relationship between cell seeding density and clinical efficacy of autologous cultured melanocyte transplantation in the treatment of vitiligo.Methods A total of 632 patients with vitiligo were enrolled in this study,and randomly classified into 4 groups to be treated with transplantation of autologous cultured melanocytes at 4 different seeding densities respectively,i.e.,(3.0-4.9)× 104/cm2 (n =201),(5.0-7.9) × 104/cm2 (n =303),(8.0-9.9) × 104/m2 (n =82),(10.0-12.0) × 104/cm2 (n =46).Epidermal sheets were obtained by suction blister biopsy from the normal skin of the vitiligo patients,and subjected to the isolation and culture of melanocytes.After 2 to 5 passages,the cultured autologous melanocytes were transplanted at different seeding densities to vitiligous lesions,which were abraded previously by ultra-pulsed CO2 laser,of these patients.All the patients were followed for 6-12 months.Results At 6 months after the transplantation,52.85％of these patients achieved more than 90％ repigmentation,and 82.28％ more than 50％ repigmentation,with no differences in the cure rate and response rate between the 4 groups (both P ＜ 0.05).The percentage of patients obtaining excellent color matching was significantly higher in the group treated with transplantation of melanocytes at a seeding density of (5.0-7.9) × 104/cm2 than in the other 3 groups at 6,12 and 24 months after treatment (all P ＜ 0.05),and higher in all the 4 groups at 12-and 24-month points compared with the 6-month point (all P ＜ 0.05),but no statistical difference was observed between the 12-and 24-month point in any of these groups (all P ＞ 0.05).Conclusions The transplantation of autologous cultured pure melanocytes is effective for the treatment of stable vitiligo with the optimal cell seeding density of melanocytes being (5.0-7.9) × 104/cm2,and the color matching appears to improve with time.