| 1. To overcome the disadvantage of traditional Tween test, we try to explore a new kind of method for Tween test, which may improve the accuracy of classification of Malassezia species. Methods First prepare four kinds of Tween cultures of different concentration gradient, then apply the suspension of Malassezia to the surface of the culture. After cultivation in 37℃for 5 days, the colony is harvested and the spores are counted under microscope. By means of variance analysis, we get the most suitable concentration of four kinds of Tween (20, 40, 60, 80) for MaIassezia. Then the new kind of Tween culture is tested by 7 type strains of Malassezia. Results 2% is the most suitable concentration of four kinds of Tween for MaIassezia. The reliability of the new method is proved by the classification of 7 type strains of Malassezia. Conclusion The new kind of Tween culture significantly improves the accuracy of classification of Malassezia species, and it also improves the efficacy and economy of the test.2. To observe the influence on the melanogenesis of B16F10 melanoma cells by the co-culture supematant of Keratinocytes with Malassezia isolates which cause hyper- or hypo- pigmentation. Methods The effects of Malassezia isolates with different proportions on the growth rate of keratinocytes Were assessed with MTT. 6 days after the B16F10 cells having been cultivated with the co-culture supernatants of different groups, the cell growth rate, the melanin content, the tyrosinase activity and the expression of tyrosinase protein of B16F10 cells are determined. Results When the ratio between keratinocytes and Malassezia isolates was lower than 1:10, the growth rate of keratinocytes was not affected by Malassezia (P＞0.05). When the ratio was increased above 1:20, the growth rate of keratinocytes was significantly inhibited by Malassezia (P＜0.01). The melanin content, the tyrosinase activity and the expression of tyrosinase protein of B16F10 cells are increased by the co-culture supematant of Keratinocytes with Malassezia isolates from hyper-pigmentation area (P＜0.01). The cell growth rate has no obvious changes (P＞0.05). The co-culture supernatant of Malassezia isolates from hoper-pigmentation area has the opposite effect. Results By the interaction with Keratinocytes, Malassezia isolates can have an influence on the tyrosinase activity and the expression of tyrosinase protein of B16F10 cells, and regulate the melanogenesis.3. To investigate the co-culture of keratinocytes with MaIassezia isolates which cause the Pityriasis Versicolor with different color and to analyze the changes of cytokines associated with melanogenesis. Methods Co-culture of keratinocytes and Malassezia species were performed with isolates from hyer-and hypo-pigmentation areas of pityriasis versicoior. The supematants were collected at different time points, and the changes of basic fibroblast growth factor (b-FGF), endothelin-1 (ET-1), nerve growth factor-β(NGF-β), interleukin-la (IL-la), interleukin-6(IL-6), tumor necrosis factor-a (TNF-a), colony stimulating factors (SCF) were recorded. Three control groups were established accordingly. Results The secretions of IL-1a, IL-6, TNF-a, and ET-1 was significantly increased after the co-culture of keratinocytes and Malassezia (P＜0.01), while those of b-FGF, NGF-β, and SCF had no significant changes (P＞0.05). Compared with the isolates from the hypo-pigmentation area, ET-1 induced by isolate from hyper-pigrnentation area significantly increased (P＜0.01). Conclusion When Malassezia isolates are co-cultured with keratinocytes, the secretions of cytokines associated with melanogenesis may differ from each other. ET-1 may play certain role in the hyper-pigmentation of pityriasis versicolor.
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