Effects Of IL-10, IL-17, IL-22and IL-23on Biological Activity Of Human Epidermal Melanocytes

Abstract:Objective:To establish a melanocyte (MC) culture system and simulate it with cytokines, then observe the effects of various cytokines on biological activity of human epidermal melanocytes including the cell proliferation, tyrosinase activity, and apoptosis of melanocytes. This will help us to study the pathogenesis of pigment aplastic skin diseases and provide new ideas for the treatment of pigment disorders. Methods:1. To establish a method of culturing melanocytes in the absence of Cholera toxin (CT),12-O-tetradecanoyl-phorbol-13-acetate (TPA) and3-isobutyl-1-methyl-xanthin (IBMX).2. Effects of IL-10, IL-17, IL-22, and IL-23on proliferative activity of human epidermal melanocytes were detected by CCK-8method respectively.3. Effects of IL-10, IL-17, IL-22, and IL-23on tyrosinase activity of human epidermal melanocytes were detected by L-DOPA method.4. Effects of IL-10, IL-17, IL-22, and IL-23on apoptosis of human epidermal melanocytes were detected by Flow cytometry method. All the data were analyzed by SPSS10.0statistics software. Results:1. By using Medium254added with HMGS-2, We could get a great quantity of pure normal melanocytes.2. With the increase of culture time, proliferation rate of cultured human epidermal melanocytes in the presence of IL-10at the concentration of50ng/mL increased. When melanocytes were cultured for48h and72h, there were statistically significant differences (P<0.01) in proliferation rate of cultured human epidermal melanocytes compared with the control group. However, with the IL-10concentration increases, the proliferation rate of melanocytes manifests a downward trend with the prolonged incubation time. When melanocytes were cultured for48h and72h, the concentration rate of melanocytes in the presence of IL-10at the concentration of200ng/ml has significant differences compared with the control group (P<0.05).3. As we prolonged the incubation time, the inhibitory ratio of melanocytes was increased in a dose-dependent manner in the presence of IL-17. Differences of inhibitory ratio of melanocytes between the IL-17groups and the control group were signicant (P<0.05) at the concentration of50ng/mL and statistically signicant (P<0.01) at the concentration of100ng/mL and200ng/mL for48h and72h.4. With the increase of concentration, the inhibitory ratio of melanocytes was increased in a time-dependent manner in the presence of IL-22. Differences of inhibitory ratio of melanocytes between the IL-22group and the control group were signicant at the concentration of200ng/ml for24h (P<0.05), at the concentration of50ng/mL,100ng/ml and200ng/ml for48h, and at the concentration of25ng/mL and50ng/ml for72h respectively. A statistically significant difference was obtained at the concentration of100ng/mL and200ng/mL (P<0.01).5. With the increase of concentration, the inhibitory ratio of melanocytes was increased in a time-dependent manner in the presence of IL-23. Differences of inhibitory ratio of melanocytes between the IL-23group and the control group were significant (P<0.05) at the concentration of200ng/mL for24h,100ng/mL and200ng/mL for48h, and50ng/mL,100ng/mL and200ng/mL for72h respectively. A statistically significant difference was obtained at the concentration of200ng/mL (P<0.01) for48h and72h.6. The detection of melanocyte tyrosinase activity:The melanocyte tyrosinase activity of IL-10groups increased and there are signicant differences between IL-10groups and the control group at the concentration of50ng/mL for48h (P<0.05). The melanocyte tyrosinase activity were inhibited in the IL-17, IL-22and IL-23group, and there were significant differences between the three groups and the control group at the concentration of50ng/mL,100ng/mL and200ng/mL for the IL-17and IL-22group,100ng/mL and200ng/ml for the IL-23group respectively.7. Melanocytes were cultured at the concentration of100ng/mL of IL-10, IL-17, IL-22, IL-23and control group for48h, the apoptosis rate of melanocytes were5.2%,32.1%,23.0%,23.0%, and5.1%respectively. No statistically significant differences were obtained between the IL-10group and the control group, while there were some differences between the IL-17, IL-22, IL-23group and control group. Conclusion:1. We get a great quantity of pure normal melanocytes by using Medium254added with HMGS-2and removing the keratinocytes and the fibroblasts according to different time of adhesion and digestion between melanocytes and keratinocytes.2. The proliferation effect of cultured human epidermal melanocytes in the presence of IL-10at the concentration of50ng/mL was increased. However, at the concentration of200ng/mL, the growth of melanocytes was inhibited instead. The effect of tyrosinase activity were consistent for both of them. The results suggested the appropriate concentration of IL-10could promote the proliferation of melanocytes and the tyrosinase activity.3. With the IL-22concentration increases, the inhibitory of proliferation of melanocytes was in a time-dependent manner. When melanocytes were cultured for72h, The proliferation of melanocytes were inhibited in IL-22groups with differences concentration. While with the concentration of IL-22increased, the tyrosinase activity of melanocytes decreased. The apoptosis rate of melanocytes increased significantly compared with the control group. The results suggest that IL-22can cause the damage of the melanocytes.4. The growth of melanocytes and the activity of tyrosinase were inhibited at the high concentration of IL-17and IL-23. As incubation time prolonged, the differences between the experimental groups and the control group became increasingly great, and the apoptosis rate of melanocytes was also greatly improved. These results suggested that the IL-17and IL-23induced injuries on melanocytes, but their mechanisms need further research and discussion.