The Study Of Tacrolimus On Melanocytes And Keratinocytes

ObjectiveTo investigate the direct and indirect effects of the new topical tacrolimus on pigmentation of melanocytes.Then get the underlying mechanism of how topical tacrolimus induces repigmentation in vitiligo.Our study provide evidence for clinical work.At the same time, topical tacrolimus could be widely used in vitiligo.Methods1.Melanogenesis in B16 melanoma cells were seeded in different culture plates for different experiments. Cells at logarithmic phase were used in these experiments.Tacrolimus was added to cultured melanogenesis in B16 melanoma cells at 0(the control group),10,102,103,104nmol/L to investigate the direct effects on them, each group was repeated three times.The measurements included cell proliferation,cell migration,tyrosinase activtion, melanin contents.2.HaCaT cells were seeded in different culture plates for different experiments. HaCaT cells at logarithmic phase were used in these experiments. Tacrolimus was added to cultured HaCaT cells at 0 (the control group),10,102,103, 104 nmol/L,followed by incubation for 48h and collection of culture supernatant,each group was repeated three times.Cytokines and growth factor in culture supernatant were measured by a commercially available enzyme-linked immunosorbent assay(ELISA) kit.The measurements included stem cell factor(SCF),basic fibroblast growth factor(bFGF). Cell proliferation was also detected. Collect HaCaT cells and melanogenesis in B16 melanoma cells,then extract RNA,investigate the level of mRNA expression of SCF and c-kit by real-time SYBR Green PCR assays.3.HaCaT cells supernatant obtained from HaCaT cells treated with various tacrolimus concentrations for 48h was added to cultured melanogenesis in B16 melanoma cells.Then investigate the.indirect effects on melanogenesis in B16 melanoma cells via HaCaT cells. The measurements included cell proliferation,tyrosinase activtion, melanin contents.Results1.Tacrolimus treatment increased tyrosinase activity and melanin contents, although there was an inhibitory effects on growth of melanocytes. Furthermore, cell migration were enhanced by tacrolimus treatment.2.Tacrolimus treatment did not increase the proliferation of HaCaT cells.The concertration of SCF in HaCaT cells supernatant increased with tacrolimus treatment. And the level of mRNA expression of SCF and c-kit were significantly impacted by tacrolimus.3.Results demonstrated that proliferation of melanogenesis in B16 melanoma cells was significantly enhanced by tacrolimus-treated HaCaT cells supernatant.But tyrosinase activity and melanin contents were not significantly impacted.ConclusionsThese findings provide evidence demonstrating direct and indirect effects of tacrolimus on pigmentation of melanocyte proliferation. Migration, tyrosinase activity,melanin contents, mRNA expression of cytokines and growth factors may provide a possible mechanism for the effect of tacrolimus in vitiligo.