白细胞介素-8对体外培养人皮肤移植物中黑素细胞增殖的影响

Effects of interleukin-8 on the proliferation of melanocytes in human skin explants cultured in vitro

目的:探究白细胞介素(IL)-8、肿瘤坏死因子(TNF)-α和IL-17A促炎因子对体外培养的皮肤移植物中黑素细胞(MC)增殖的影响。方法:建立气-液界面培养人皮肤移植物模型,采用常规苏木精-伊红(HE)染色观察培养后不同时间(第0、2、3、5天)人皮肤移植物组织结构的完整性和细胞存活状态。将皮肤组织分为对照组、IL-8组、TNF-α+IL-17A组和TNF-α+IL-17A+IL-8组,培养48 h后,采用HE染色观察表皮的增殖与分化;采用抗HMB45抗体免疫荧光染色观察MC数目;采用CCK8细胞增殖检测试剂盒测定MC的增殖率。结果:皮肤移植物在气-液界面培养第5天,表皮与真皮界面仍保持其完整性,且未见表、真皮分离。与对照组相比,TNF-α+IL-17A组皮肤移植物的角质层明显增厚(P<0.05),且可明显观察到角化不全,与银屑病皮损组织病理改变相似。IL-8组和TNF-α+IL-17A+IL-8组中HMB45阳性细胞数目均较对照组明显增加,差异均有统计学意义(P<0.05)。采用体外培养的PIG1细胞验证IL-8对MC的促增殖作用,结果示与对照组比较,IL-8组中MC细胞增殖率差异无统计学意义(P>0.05)。结论:银屑病相关促炎因子TNF-α及IL-17A对IL-8促MC增殖存在协同刺激作用。

Objective:To investigate the effects of proinflammatory cytokines,such as interleukin(IL)-8,tumor necrosis factor(TNF)-α and IL-17A,on the proliferation of melanocytes(MCs)in human skin explants cultured in vitro.Methods:Full thickness human abdominal skin explants were cultured on Transwell inserts via an air-liquid interface(ALI)culture.After culture,the tissue structure and cell morphology of human skin explants were examined at different time points(days 0,2,3,5) using conventional HE staining.The skin explants were divided into four groups:control group,IL-8 group,TNF-α+IL-17A group and IL-8+TNF-α+IL-17A group.After 48 h of treatment,the proliferation and differentiation of the skin explants were observed using HE staining.The number of melanocytes in the explants was visualized with anti-HMB45 monoclonal antibody by immunofluorescent staining.Lastly,the proliferation of human melanocyte PIG1 treated with IL-8 alone or combined with TNF-α and IL-17A was examined using the cell counting kit-8(CCK8) assay.Results:The interface between the epidermis and dermis of skin explants cultured on Transwell inserts remained intact until day 5 after ALI culture.No disconnection between the epidermis and dermis was observed.Compared with the control group,the stratum corneum of skin explants treated with the combination of TNF-α with IL-17A thickened significantly(P0.05).Conclusion:Psoriasis-associated proinflammatory cytokines TNF-α and IL-17A have a synergistic effect on IL-8-induced melanocyte proliferation.