Study On The Effects Of Malassezia On Metabolizing And Tyrosinase Gene Expression Of Human Melanocytes

[Objective]: The primary lesion of pityriasis versicolor caused by Malassezia is hypopigment and /or hyperpigment. Are the pigmentary changes connected with the biology difference of different Malassezia species in addition to considering individual difference? In this study, we use seven species of Malassezia, to react with melanocytes, by directly and indirectly, with the condition medium after co-culture with keratinocyte, to investigate the effects of Malassezia on tyrosinase activity, melanogenesis and tyrosinase gene expression of human melanocytes.[Methods]: Normal human melanocytes were isolated from healthy men and cultured with MCDB153 medium supplemented with 12-O-tetradecanoyl phorbol-14-acetate (TPA), cholera toxin (CT), recombinan human epidermal growth factor (rhEGF) and isobutyl methylxanthine (IBMX). Dopa stain, Fontana silver stain and S-100 protein histochemical stain were performed to identify the cultured melanocytes cells. The reaction assay system of tyrosinase activity was established according to the principle of oxidation. Co-culture supernatants were obtained after 24 hours of culturing keratinocytes with different Malassezia. The medium used for melanocyte experiments was 1:1, 1:3 and 1:7 (v:v) mixture of co-culture supernatants andmelanocyte growth medium. Dopa oxidization reaction and NaOH assay were used to determine the activity of tyrosinase and synthesis of melanin; MTT method was used to determine the proliferation of melanocytes; Real-Time PCR assay was used to determine the tyrosinase gene expression of human melanocytes. The ultra structural changes of melanocytes were observed by transmission electron microscopy(TEM). [Results]: 1. The melanocytes culture system is stable and melanocytes could be cultured for five passages in vitro. It is proved to be melanocytes by the dopa stain, Fontana silver stain and S-100 protein histochemical stain. 2. The reaction system of tyrosinase activity is stable and credible. The optimal reation buffer was pH 9.0, 0.25 mol/L Tris-HCl; The optimal concentration of substrate L-Dopa was 0.15% and dissolved with pH 8.0, 0.01 mol/L Tris-HCl; The sensitivity of reation was increased markedly when affiliate 0.03% H2O2 in substrate; The optimal melanocytes amount of test was ≥ 2.5 ×104, the reation fastigium was within twenty minutes and connected with the melanocytes amounts, the more of the melanocytes number, the earlier of fastigium. 3. Increased tyrosinase activity (A450 value increasing from 1.298 ± 0.046 to 1.559 ± 0.059 per minute) and melanogenesis (increasing from 6.593 ± 0.852 mg/L to 13.715 ± 0.811 mg/L) are showed in melanocytes treated 24 hours with culture supernatants of co-culture of keratinocytes with Malassezia globosa compared with non-treated melanocytes; Increased tyrosinase gene expression (the relative fluorescence intensity ratio of Real Time RT-PCR product of tyrosinase mRNA to comparison GAPDH mRNA increasing from 0.911 ± 0.562 to 35.445 ± 26.297) of human melanocytes are showed in melanocytes treated with culture supematants of co-culture ofkeratinocytes with Malassezia globosa compared with non-treated melanocytes; Abundant mitochondria, endoplasmic reticulum, ribosome, and melanin granule were shown in cell plasma under TEM. 4. There is no difference of tyrosinase activity, melanogenesis, and tyrosinase gene expression when melanocytes are treated 24 hours with culture supernatants of co-culture of keratinocytes with the other six species of Malassezias compared with non-treated melanocytes. 5. There is no difference of tyrosinase activity in melanocytes treated 24 hours directly with all of the seven species Malassezia compared with non-treated melanocytes. [Conclusions]: 1. The co-culture supernatants of keratinocytes with Malassezia globosa could increase tyrosinase activity, melanogenesis, tyrosinase gene expression of melanocytes. Abundant mitochondria, endoplasmic reticulum, ribosome and melanin granule were shown in cell plasma. 2. There is no difference of tyrosinase activity, melanogenesis, and tyrosinase gene expression when melanocytes are treated 24 hours with culture supernatants of co-culture of keratinocytes with the other six species of Malassezias compared with non-treated melanocytes. 3. There is no change of tyrosinase activity in melanocytes treated 24 hours directly with Malassezia.