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Objective To evaluate the effect of latanoprost on cell proliferation of and melanogenesis in human epidermal melanocytes,and to explore its mechanism.Methods Latanoprost was added into the 254 medium to prepare latanoprost solutions at different concentrations of 10-5,10-6 and 10-7 mol/L.In vitro cultured human epidermal melanocytes were divided into 4 groups to be cultured with media containing no latanoprost (control group) or 10-5,10-6 and 10-7 mol/L latanoprost for 48 hours.Cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative activity of melanocytes,dopa oxidation assay to estimate the activity of tyrosinase.Sodium hydroxide (NaOH)-lysis method was used to determine the content of melanin,and Masson-Fontana staining to observe the number and distribution of melanin granules.Westernblot analysis and real-time fluorescence-based quantitative PCR were performed to determine the protein and mRNA expression of melanogenesis-related genes including microphthalmia-associated transcription factor (MITF),tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1).Comparison among the 4 groups and multiple comparisons were done by using one-way analysis of variance and least significant difference (LSD)-t test.Results Compared with the control group,the 10-6-,10-5-mol/L latanoprost groups showed significantly increased proliferative activity of melanocytes (1.064 ± 0.172 and 1.078 ± 0.080 vs.0.784 ± 0.015;t =3.289,3.454 respectively,both P ＜ 0.05),increased activity of tyrosinase (0.510 ± 0.017 and 0.454 ± 0.009 vs.0.355 ± 0.041;t =6.139,3.939 respectively,P ＜ 0.01 or 0.05),and increased content of melanin (t =7.232,5.967,both P ＜ 0.01).However,there were no significant differences in the proliferative activity of melanocytes,activity of tyrosinase or content of melanin between the 10-7-mol/L latanoprost group and control group (all P ＞ 0.05).Masson-Fontana staining showed more and darker melanin granules on melanocyte dendrites in the 10-5-,10-6-,10-7-mol/L latanoprost groups than in the control group,and the color of melanin granules changed from light brown to black brown along with the increase in the concentration of latanoprost.The mRNA expression of MITF increased along with the increase in the concentration of latanoprost (P ＜ 0.01),and the protein expression of MITF wassignificantly higher in the 10-6,10-5-mol/L latanoprost groups than in the control group and 10-7-mol/L latanoprost group (all P ＜ 0.01).The 10-6-mol/L latanoprost group showed significantly increased mRNA and protein expression of TYR and TYRP1 compared with the control group,10-7-,10-5-mol/L latanoprost groups (all P ＜ 0.01).Conclusion Latanoprost can increase the proliferation of human epidermal melanocytes,and promote tyrosinase activity and melanogenesis likely by enhancing the mRNA and protein expression of MITF,TYR,TYRP1.
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Objective To investigate relationships between serum levels of anti?tyrosinase IgG antibody(TYR IgG)as well as anti?tyrosinase?related protein?1 IgG antibody(TRP?1 IgG)and vitiligo. Methods Enzyme linked immunosorbent assay(ELISA)was performed to detect serum levels of TYR IgG and TRP?1 IgG in 260 patients with vitiligo and 50 health controls. The threshold for defining a positive test result was set at 3 standard deviations above the mean serum level of TYR IgG or TRP?1 IgG in the healthy controls. Results The positive rate of TYR IgG and/or TRP?1 IgG in the vitiligo group was 57.31%(149/260). The positive rates of TYR IgG and TRP?1 IgG were both significantly higher in the vitiligo group than in the control group(TYR IgG:37.3%[97/260]vs. 0,χ2=25.441, P0.05). Among patients with vitiligo, the positive rate of TRP?1 IgG was significantly higher in females than in males(χ2=5.811, P20 years(χ2=6.498, P 20 years (both P >0.05). Conclusion Detection of TYR IgG and TRP?1 IgG may provide some evidence for the diagnosis and treatment of vitiligo.
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Objective To study to the effect of 1064-nm Q-switched Nd:YAG laser irradiation on the melanogenesis in a human epidermal melanocyte line PIG. Methods Cultured PIG cells were irradiated with 1064-nm Q-switched Nd:YAG laser (Medlite C6) at different energy densities for 10 times. After additional culture for various durations, cell viability was detected by MT assay, tyrosinase activity by dopa oxidation assay, mRNA and protein expressions of tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2 by real-time quantitative fluorescent RT-PCR and Westen blotting respectively, Results The irradiation with Q-switched Nd:YAG laser at energy densities from 1 to 3 J/cm2 had no obvious effect on the viability of PIG cells. After irradiation with Nd:YAG laser at 1 J/cm2, PIG cells showed a significant increase in the tyrosinase activity,mRNA expressions of tyrosinase and TRP-1 compared with unirradiated cells (0.563 ± 0.014 vs 0.501 ±0.019, 1.40±0.11 vs 1.0, 1.28 ± 0.03 vs 1.0, all P＜ 0.05), but both the mRNA (0.91 ± 0.17 vs 1.0, P＞0.05) and protein expressions of TRP-2 experienced no significant changes before and after the irradiation.However, a significant decrease was noted in PIG cells irradiated with Nd:YAG laser at 3 J/cm2 in tyrosinase activity, mRNA and protein expressions of tyrosinase (0.70 ± 0.02 vs 1.0, 0.64 ± 0.05 vs 1.0, both P ＜ 0.05),TRP-1 (0.73±0.04 vs l.0, 0.86±0.17 vs l.0, both P＜0.05) andTRP-2 (0.68±0.04 vs l.0,0.69±0.11vs 1.0, both P ＜0.05) in comparison with unirradiated PIG cells. Conclusions The 1064-nm Q-switched Nd:YAG laser irradiation may affect the melanogenesis in PIG cells. With no influence on cell viability, the 1064-nm Q-switched Nd:YAG laser at 1 J/cm2 could enhance melanogenesis, while that at 3 J/cm2 could suppress melanogenesis, in PIG cells.
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Objective: To study the inhibition of antisense TRP1 on cell growth of malignant melanoma(MM) and explore a new way for therapy of melanoma. Methods: Antisense TRP-1 recombinant vector was constructed and transfected into MM cells. According to the results of MTT, cell growth curves were drawn and then clonogenic assay was performed in vitro. At last, tumorigenesis assay was undertaken in nude mice in vivo. Results: Cell proliferations of TRP-1 transfected MM cells were inhibited compared with the control cells. The results of clonogenic assay displayed the difference of clonogenic percentage between TRP-1 transfected MM cells (52% , P
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Objective To construct the antisense eukaryotic vector of human TRP-1 (tyrosinase related protein 1) encoding gene, and transfect it into TRP-1 highly expressed melanocytes and malignant melanoma cell line, in order to further study the effects of antisense TRP-1 on the proliferation and functions of those cells. Methods TRP-1 cDNA was amplified by polymerase chain reaction (PCR) and the PCR products were subcloned into eukaryotic expression vector pcDNA3.1 on the opposite direction. Antisense recombinant vector was transfected into melanocytes and melanoma cell line. TRP-1 mRNA level was detected by reverse transcriptase polymerase chain reaction (RT-PCR). TRP-1 protein level was detected by Western blot. Cell cycle was determined by flow cytometry. The activity of tyrosinase was valued by L-dopa reaction. Results The recombinant antisense vector pcDNA3.1/TRP-1(-) was constructed. Positive transfected cells could steadily express TRP-1 antisense RNA. It was showed that there was a low level of TRP-1 mRNA as indicated by RT-PCR, and a low level of TRP-1 protein as indicated by Western blot. Cell cycles were blocked in G1 stage. The suppress rates of tyrosinase was 46% in transfected melanocytes and 54% in malignant melanoma cells, respectively. Conclusions TRP-1 plays an important role in the proliferation and functions of melanocytes and melanoma cells. Antisense TRP-1 could block the cell cycles and decrease the activity of tyrosinese in those cells.
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Objective To modify HLA-A2.1 restricted epitope of tyrosinase related protein-2 (TRP-2) (180-188) of human melanoma differentiation antigen and enhance HLA-A2.1 molecule its affinity to. Methods The TRP-2 (180-188) nave epitope was altered with the predominant amino acid for the primary anchor residues and auxiliary anchor residues. Altered peptide ligands (APLs) were scanned by polynomial method, the quantitative motif method and OSAR methods. The analogues their affinity to HLA-A2.1 molecule were assayed. Results Compared with the nave peptide, APLs had stronger affinity to HLA-A2 molecules. Conclusion Our results suggest that altered nave epitope with P1 (Y) and/or P2(L, M) can enhance affinity to HLA-A2 molecule. However, the immunogical efficacy of APLs needs to be identified in studies in vitro and in vivo.