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Annals of Dermatology ; : 553-561, 2021.
Article in English | WPRIM | ID: wpr-913450


Background@#Androgenetic alopecia (AGA) leads to thinning of scalp hair and affects 60%~70% of the adult population worldwide. Developing more effective treatments and studying its mechanism are of great significance. Previous clinical studies have revealed that hair growth is stimulated by 650-nm red light. @*Objective@#This study aimed to explore the effect and mechanism of 650-nm red light on the treatment of AGA by using ex vivo hair follicle culture. @*Methods@#Human hair follicles were obtained from hair transplant patients with AGA. Hair follicles were cultured in Williams E medium and treated with or without 650-nm red light.Real-time RT-PCR and immunofluorescence staining were used to detect the expression level of genes and proteins in hair follicles, respectively. RNA-sequencing analysis was carried out to reveal the distinct gene signatures upon 650 nm treatment. @*Results@#Low-level 650 nm red light promoted the proliferation of human hair follicles in the experimental cultured-tissue model. Consistently, 650 nm red light significantly delayed the transition of hair cycle from anagen to catagen in vitro. RNA-seq analysis and gene clustering for the differentially expressed genes suggests that leukocyte transendothelial migration, metabolism, adherens junction and other biological process maybe involved in stimulation of hair follicles by 650-nm red light treatment. @*Conclusion@#The effect of 650-nm red light on ex vivo hair follicles and the transcriptome set which implicates the role of red light in promoting hair growth and reversing of miniaturization process of AGA were identified.

Chinese Journal of Dermatology ; (12): 302-309, 2019.
Article in Chinese | WPRIM | ID: wpr-745785


Objective To evaluate the inductive effect of interferon-γ(IFN-γ) combined with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on programmed necrosis of the human immortalized keratinocyte cell line HaCaT,and to explore its mechanisms.Methods In vitro cultured HaCaT cells were divided into several groups:negative control group receiving no treatment,IFN-γ group treated with 50 μg/L IFN-γ,TRAIL group treated with 4 μg/L TRAIL,and cytokine combination group treated with 50 μg/L IFN-γ and 4 μg/L TRAIL or zVAD combination group pretreated with 40 μmo/L zVAD for 1 hour followed by the treatment with 50 μg/L IFN-γand 4 μg/L TRAIL.After 48-hour treatment,the morphology of HaCaT cells were observed under a light microscope,methyl-thiazolyl-tetrazolium assay was performed to evaluate the inhibitory effect of the treatment on the proliferation of HaCaT cells,and double staining flow cytometry to detect the necrosis of HaCaT cells.Meanwhile,real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA expression of receptor interaction protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL),Western blot analysis to determine the expression of RIP1,RIP3,MLKL proteins and their phosphorylated forms (pRIP1,pRIP3,pMLKL),immunofluorescent staining to observe the distribution of pRIP3 and pMLKL in HaCaT cells,and the level of reactive oxygen species (ROS) in HaCaT cells in the above groups was detected by the fluorescence probe DCFH-DA.Statistical analysis was carried out with SPSS 22 software by using one-way analysis of variance (ANOVA) for comparing indices among different groups,and least significant difference (LSD)-t test for multiple comparisons.Results After 48-hour treatment,HaCaT cells in the cytokine combination group and zVAD combination group showed necrosis-like morphologic features.Methyl-thiazolyl-tetrazoliumassay revealed significant differences in the survival rate of HaCaT cells among the IFN-γgroup,TRAIL group,cytokine combination group,zVAD combination group and negative control group (73.16% ± 5.71%,81.46% ± 4.68%,72.18% ± 2.93%,69.67% ± 3.24% and 100%,respectively;F =24.34,P < 0.001).The necrosis rate of HaCaT cells was notably higher in the cytokine combination group and zVAD combination group (9.86% ± 1.31%,10.33% ± 2.16%,respectively) than in the negative control group (5.26% ± 0.91%,t =4.61,5.07,respectively,both P < 0.05).qPCR revealed that the mRNA expression of RIP3 and MLKL significantly increased in the cytokine combination group and zVAD combination group compared with the negative control group (tRIP3 =0.99,1.84,tMLKL =1.51,2.17,respectively,all P < 0.05).Western blot analysis suggested that the protein expression of RIP1,RIP3,MLKL,pRIP1,pRIP3 and pMLKL significantly increased in the cytokine combination group compared with the negative control group (all P < 0.05),and the zVAD combination group showed significantly decreased caspase 8 expression and increased expression of the above proteins compared with the cytokine combination group.Fluorescence microscopy showed that enhanced green dot-like or clump-like fluorescent spots (representing pRIP3) could be observed in the cytoplasm,and red fluorescent spots (representing pMLKL) could be seen on the cell membrane in the cytokine combination group.The average fluorescence intensity of ROS was significantly higher in the cytokine combination group than in the negative control group (t =702.00,P < 0.05).Conclusion IFN-γcombined with TRAIL can induce the programmed necrosis of HaCaT cells with increased level of ROS.

Article in Chinese | WPRIM | ID: wpr-405732


Objective To express, purify and identify tetrameric protein of methyl-CpG-binding domain in E. coli. Methods The recombinant plasmid 4 × MBD-pET30b + were transformed into E. coli DH5a for clonal expansion and sequenced, then the tetrameric-proteins were expressed in E. coli BL21 (DE_3) under the induction of IPTG. Moreover, the expression products were purified by Ni-NTA chromatography, and were determined by SDS-PAGE and Western blot. Immunostain 293T cells with the proteins were analyzed by fluorescence microscope. Results The sequence analysis showed orientation right and was identical with the expectation. SDS-PAGE and Western blot demonstrated that the molecular weight of the tetrameric- protein was 46 0110 with the N-terminal His-tag and the C-terminal HA-tag. The MBD proteins can bind to the intracellular CpG DNA specifically.Conclusions The tetrameric-proteins of methyl-CpG-binding domain are successfully expressed and purified in E. coli. This results establish a groundwork for the further researches on DNA methylation.

Chinese Journal of Dermatology ; (12): 800-802, 2008.
Article in Chinese | WPRIM | ID: wpr-397552


The patient was a 38-year woman.Six years prior to presentation,she developed erythema and papules with occasional pruritus in both labium majora,which gradually confluenced into plaques with the formation of superficial erosion and ulcer;extension into the vulva and crissum occurred later.Half a year prior to presentation,a subcutaneous firm nodule measuring about 1.5cm in diameter with intact epidermis,developed on the right submaxilla,associated with mild swelling in the area;the nodule enlarged gradually.A subcutaneous induration measuring 0.5 cm in diameter was observed in the left chin 2 months later.She also reported a 20-year history of dry eye and dry mouth.The patient tested postive for antinuclear antibody cally,there was a focal infiltration of lymphocytes in tissue of minor salivary glands.The pathology of lesions on the right labium majus showed a dense dermal infiltration with S100-and CD1a-positive,irregulady-shaped histiocyte-like cells with abundant eosinophilic cytoplasm and few mitotic figures.Needle biopsy of the right submaxilla area showed tumor cells.A diagnosis of Langerhans cell histiocytosis and Sj(o)gren syndrome was made based on clinical manifestation,laboratory findings and histopathological features.Combination chemotherapy with cyclophosphamide,vincristine,corticosteroids and etoposide resulted in clinical improvement.