马拉色菌在花斑癣色素改变中的作用

目的 观察引起不同临床色素表现的马拉色菌与角质形成细胞（KC）共培养液对B16F10黑素瘤细胞黑素合成的影响，及共培养液中与黑素合成相关的细胞因子产生情况。方法 将1 × 106/mL KC与分离自色素沉着区的马拉色菌（简称C株）、分离自色素减退区的马拉色菌（简称J株）按4个浓度比例（KC ∶ 马拉色菌为1 ∶ 1、1 ∶ 10、1 ∶ 20、1 ∶ 30）分别共培养，用MTT法测定KC增殖率。将C株或J株马拉色菌分别加入KC培养体系共培养，真菌孢子和KC密度均为1 × 106/mL（1 ∶ 1），制备的培养液分5组，KC单独培养液、C株或J株马拉色菌与KC共培养液、C株或J株马拉色菌单独培养后再次培养KC的二次培养液，用上述各组培养液分别培养B16F10细胞，用MTT法测量各组B16F10细胞增殖率，NaOH裂解法测量黑素含量，Western印迹法测定酪氨酸酶和β-肌动蛋白的表达，多巴氧化法测定酪氨酸酶活性。ELISA法测定各组培养液中细胞因子的浓度。结果 KC与马拉色菌在1 ∶ 10比例以下，KC的生长不受马拉色菌的影响。C株马拉色菌与KC共培养液引起B16F10细胞黑素含量、酪氨酸酶活性和酪氨酸酶蛋白表达增加，细胞增殖率无明显变化。J株马拉色菌共培养液的作用与之相反。C株马拉色菌刺激产生的内皮素1显著高于J株马拉色菌（P < 0.01）。结论 马拉色菌通过与KC相互作用，影响B16F10细胞酪氨酸酶的活性和表达，调节黑素合成。内皮素1在花斑癣色素沉着中可能起一定作用。

Role of Malassezia in the color diversity of pityriasis versicolor

Objective To investigate the influence of Malassezia isolates that can cause hyperpigmentation or hypopigmentation in patients with pityriasis versicolor on the melanogenesis of B16F10 melanoma cells.and to detect the levels of cytokines associated with melanogenesis.Methods Primary culture of keratinocytes(1×106 cells/mL)derived from human prepuce were co-cultured with Malassezia strains isolated from hyperpigmented or hypopigmented lesions in the same patient with pityriasis versicolor,with the concentration ratio of keratinocytes to Malassezia cells being 1:1,1:10,1:20 and 1:30,respectively.The proliferation rate of keratinocytes was detected by MTT methOd.To observe the influence on B16F10 melanoma cells.five difierent types of culture supematant were prepared,i.e.the supematant from culture system of keratinocytes alone,supematant from the coculture system of keratinocytes and Malassezia isolates from hyperpigmented sites or hypopigmented sites,and supernatant from keratinocyte culture in the presence of Malassezia cell-free supematant from the culture medium of Malassezia strains from hyperpigmented or hypopigmented sites.ELISA test was performed in these supematants to detect the levels of several eytokines associated with melanogenesis,including basic fibroblast growth factor(b-FGF),endothelin-1,nerve growth factor(NGF)-β,IL-1α,lL-6,tumor necrosis factor(TNF)-α and stem cell factor(SCF).B16F10 cells were cultured with the above 5 supematants.After 48-hour culture.the proliferation rate of B16F10 cells was measured with MTT method,melanin content by NaOH assay.expression and activity of tyrosinase via Western blot and dopa-oxidase assay,respectively.Results The growth rate of keratinocytes was not affected by MalassczJa(P＞0.05)when the concentration ratio of keratinocytes to Malassezia cells was more than 1:20.The melanin content,tyrosinase activity and expression of tyrosinase of B16F10 cells were inereased in the presence of supernatant from co-culture of keratinocytes with Malassezia isolates from hyperpigmentated sites (P＜0.0 1).while the supematant from the co-culture of keratinocytes with Malassezia isolates from hypopigmentated sites showed the opposite effects on the above parameters.The proliferation rate of B16F10 cells had no obvious changes(P＞0.05)in the presence of supernatant from co-culture system.The level of endothelin-1 in the coculture supematant of keratinocytes with Malassczia isolates was significantly higher than that in the culture supematant of keratinocytes alone (P＜0.01).Malassezia isolates from hypopigmented sites induced a higher level of secretion of endothelin-1 than those from hyperpigmented sites(P＜0.01).Conclusions Malassezia isolates may influence the activity and expression of tyrosinase in B16F10 cells via the interaction with keratinocytes.resulting in subsequent regulation of the melanogenesis in these cells.Endothelin-1 might play a role in the hypcrpigmentation caused by Malassezia in pityriasis versicolor.