Objective:To evaluate the effect of nicastrin (nct) gene on the biological functions of melanocytes in zebrafish.Methods:By using a morpholino oligonucleotide (MO) technology, a nct-MO sequence targeting the zebrafish nct mRNA was designed, so was a MO control (ctrl-MO) sequence. Then, the enhanced green fluorescent protein (EGFP) mRNA with MO target sequence at its 5′ end was synthesized, and co-microinjected with the nct-MO or ctrl-MO sequence into the zebrafish embryos to verify the silencing efficiency of nct-MO and observe changes in developmental phenotypes in zebrafish. With wild-type zebrafish as a blank control group, real-time fluorescence-based quantitative PCR (RT-PCR) was conducted to determine the mRNA expression of melanin synthesis-and notch signaling pathway-related genes, including mitfa, tyr, tyrp1a, tyrp1b, dct, pmela, notch1a, notch1b and hey1 genes. One-way analysis of variance was used for the comparison of means among multiple groups, and least significant difference (LSD) - t test for multiple comparisons. Results:Eight hours after zebrafish fertilization, green fluorescence was observed in the zebrafish embryos in the ctrl-MO+EGFP mRNA group, but not in the nct-MO+EGFP mRNA group or blank control group. Forty-eight hours after fertilization, the proportion of pigmented area among the whole area of the tail of zebrafish larvae was significantly lower in the nct-MO group (0.169 ± 0.083) than in the ctrl-MO group (0.258 ± 0.042, t=3.202, P=0.005) , and disorderly pigment distribution in the tails was observed in the nct-MO group. RT-PCR revealed significant differences in the mRNA expression of pmela, tyrp1a and hey1 genes among the nct-MO group, ctrl-MO group and blank control group (all P < 0.05) , but no significant difference was observed in the mRNA expression of mitfa, tyr, tyrp1b, dct, notch1a or notch1b genes among the 3 groups (all P>0.05) ; the relative expression levels of pmela and tyrp1a mRNAs were significantly lower in the nct-MO group (0.708 ± 0.028, 0.558 ± 0.136, respectively) than in the ctrl-MO group (1.023 ± 0.142, 1.016 ± 0.134, respectively, both P < 0.05) . Conclusion:The nct gene may affect biological functions of melanocytes by regulating melanin synthesis in zebrafish.

Objective:To establish a presenilin enhancer-2 (PSENEN) gene-silenced human immortalized keratinocyte (HaCaT) cell model, and to evaluate the effect of PSENEN gene silencing on the proliferation of and γ-secretase expression in HaCaT cells.Methods:Three shRNAs targeting the PSENEN gene were constructed, and inserted into the linearized LV3-pGLV-h1-GFP-puro vector to establish a recombinant lentiviral expression plasmid. After restriction enzyme digestion and sequencing, lentiviral packaging and purification were performed, and lentiviral titer was determined. Cultured HaCaT cells were divided into 5 groups: shRNA1, shRNA2 and shRNA3 groups treated with the lentivirus solutions containing PSENEN gene-targeted shRNA1, shRNA2 and shRNA3 respectively, NC group treated with the lentivirus solution containing a negative control shRNA (shNC) , and blank group treated without lentivirus solution. After transfection, inverted fluorescence microscopy was performed, and transfection efficiency was determined by flow cytometry. Cell counting kit-8 (CCK8) assay was performed to evaluate the effect of PSENEN gene silencing on the proliferation of HaCaT cells, and real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were conducted to determine the mRNA and protein expression of PSENEN, nicastrin (NCT) , presenilin-1 (PS1) and anterior pharynx defective 1a (APH1a) genes respectively. Statistical analysis was carried out by using repeated measures analysis of variance, one-way analysis of variance, and least significant difference t test for multiple comparisons. Results:Inverted fluorescence microscopy showed that fluorescence was observed in the shRNA1 group, shRNA2 group, shRNA3 group and NC group, and flow cytometry showed that the transfection efficiency was over 98% in the above 4 groups. qPCR and Western blot analysis revealed that the mRNA and protein expression of PSENEN gene significantly decreased in the shRNA1 (0.187 ± 0.010, 0.219 ± 0.097, respectively) , shRNA2 (0.163 ± 0.022, 0.208 ± 0.014, respectively) and shRNA3 (0.174 ± 0.009, 0.185 ± 0.062, respectively) groups compared with the NC group (1.054 ± 0.272, 1.076 ± 0.075, respectively, all P < 0.001) . CCK8 assay showed that the cellular proliferative activity significantly increased in the shRNA1 group compared with the NC group at 0, 12, 36 and 48 hours (all P < 0.05) , and there was no significant difference between the 2 groups at 24 or 60 hours (both P > 0.05) ; the cellular proliferative activity was significantly higher in the shRNA2 and shRNA3 groups than in the NC group at 0, 12, 24, 36, 48 and 60 hours (all P < 0.05) . There was no significant difference in the mRNA expression of NCT, PS1 and APH1a genes among the shRNA1 group, shRNA2 group, shRNA3 group, NC group, and blank group ( F= 8.168, 4.644, 1.981, respectively, all P > 0.05) , while the relative protein expression level of mature NCT (mNCT) , immature NCT (imNCT) , carboxyl-terminal fragment of PS1 (PS1-CTF) and APH1a significantly differed among the above 5 groups ( F= 39.268, 5.929, 27.842, 20.663, respectively, all P ≤ 0.01) . Compared with the NC group, the shRNA1, shRNA2 and shRNA3 groups all showed significantly decreased protein expression of mNCT, PS1-CTF and APH1a (all P < 0.01) , but insignificant changes in imNCT protein expression (all P > 0.05) . Conclusion:The PSENEN gene-silenced HaCaT cell model was successfully constructed, and the PSENEN gene silencing could lead to an increase in the cellular proliferative activity of HaCaT cells and a decrease in the protein expression of γ-secretase subunits mNCT, PS1-CTF and APH1a.

Objective To construct a lentiviral vector delivering the Nicastrin (NCT) gene-targeted short hairpin RNA (shRNA) and determine gene-silencing efficiency of the vector in the human immortalized keratinocyte cell line HaCaT,and to construct a NCT gene-silenced HaCaT cell model to lay an experimental foundation for subsequently studying effects of NCT gene silencing on biological behavior of keratinocytes.Methods Three NCT gene-targeted shRNAs were designed and inserted into the pGLV3/ H1/GFP + Puro vector to construct three recombinant plasmids,which were then confirmed by sequencing.Recombinant plasmids combined with lentivirus packaging plasmids were co-transfected into 293T cells to obtain lentivirus particles,and the virus titer was determined.Cultured HaCaT cells were divided into 3 groups:blank group receiving no treatment,negative control group infected with the empty vector LV3-shNC,interference groups infected with lentivirus NCT-shRNA1,-shRNA2,-shRNA3,respectively.Flow cytometry was performed to determine transfection efficiency,and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine efficiency of target gene silencing in HaCaT cells,so as to select the most efficient interference sequence.Results Sequencing analysis indicated that recombinant lentiviral vector NCT-shRNA was constructed successfully.After co-transfection of recombinant plasmids and lentivirus packaging plasmids into 293T cells,the titer of recombinant lentivirus particles was about 109 TU/ml.Flow cytometry showed that the transfection efficiency was greater than 95％.qRT-PCR revealed that the NCT mRNA expression was obviously down-regulated in the interference group compared with the negative control group,and NCT-shRNA1 was the most efficient sequence with interference efficiency being 75％.Western blot analysis showed that the inhibition rate of NCT protein expression was 71.7％ in the shRNA1 group compared with the negative control group.Conclusion The most efficient NCT-shRNA interference sequence is screened out,and the recombinant lentiviral vector NCT-shRNA and an NCT gene-silenced HaCaT cell model are both constructed successfully.

In this review, guidelines and consensus for the diagnosis and treatment of vitiligo in Europe, the United States, Japan, South Korea, China and other countries and regions were compared in terms of clinical typing, staging, severity assessment, drug treatment and non-drug treatment of vitiligo, and similarities and differences in recommended treatments of vitiligo as well as emphasized concepts among these guidelines and consensus were summarized. It is hoped that this review will help clinicians provide more appropriate individualized treatment for patients with vitiligo.

Objective:To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures (Dowling-Degos disease, DDD) .Methods:HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats (CRISPR) -CRISPR-associated protein 9 (Cas9) technology (experimental group) , and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn) separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 (Pmel17) in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results:Sanger sequencing showed a heterozygous mutation (c.1delA) at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group (0.60 ± 0.05) than in the control group (1.00 ± 0.00, t = 32.38, P = 0.001) . Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% ( t = -3.48, P = 0.025) in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion:The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.

By bioinformatics analysis, a putative keratinase gene gm2886 (Accession number: KY368946) was discovered in the genome of a feather-degrading strain, Streptomyces albidoflavus Fea-10. gm2886 was ligated into integrative Escherichia coli-Streptomyces shuttle vector pSET152 under the promoter PermE and added with C-terminal His-tag. The expression vector was transformed into Streptomyces pactum ACT12 by conjugal transfer and the recombinant protein GM2886-His6 was detected in fermentation broth. GM2886-His6 was purified and characterized. Its size was nearly 36 kDa. GM2886-His6 showed proteolytic activity towards a variety of substrates and could even degrade insoluble substrates, such as azure keratin and chicken feathers. The optimal pH and temperature of GM2886-His6 for proteolysis of casein was pH 10.0 and 50 ℃, respectively. The enzyme activity was inhibited by PMSF, but not EDTA, indicating that GM2886-His6 was a serine proteinase. Our results laid the foundation for the research of the molecular biological mechanism on feather-degrading and for the further utilization of Fea-10.