角质形成细胞对胞内金黄色葡萄球菌抗菌作用研究

Antibacterial effect of human epidermal keratinocytes against intracellular Staphylococcus aureus

目的:观察金黄色葡萄球菌在人角质形成细胞株HaCaT中生存的动态变化,了解金黄色葡萄球菌与皮肤角质形成细胞之间的相互关系。方法:用金黄色葡萄球菌标准株ATCC25923侵袭HaCaT细胞,分别于细菌进入细胞后的4、24、48、72h裂解细胞,释放出细胞内的活细菌,用平板菌落计数法计数胞内活菌。结果:金黄色葡萄球菌ATCC25923株在进入HaCaT细胞的24h有一定生长,但实验48h细胞内活菌数量明显减少。蛋白激酶C激活剂PMA和腺苷酸环化酶激活剂FSK可以促进HaCaT细胞清除胞内细菌。结论:皮肤角质形成细胞清除进入细胞内的金黄色葡萄球菌,可能是皮肤天然免疫的一种防御机制,而PMA和FSK增强细胞的抗菌作用,提示角质形成细胞抗菌活性与NADPH氧化酶相关。

Objective： To further understand the host-pathogen interaction in the human skin, the dynamic survival of Staphylococcus aureus in the human epidermal keratinocytes was analyzed. Methods：The monolayers of human keratinocyte cell line HaCaT were incubated with Staphylococcus aureus ATCG29523 for 2h. After the extracellular bacteria were killed with gentamicin, HaCaT ceils were lysed at different time points from 4 hours to 72 huors with Triton X-100 and viable intracellular bacteria were quantified by quantitative colony counting method. Results.. Compared with the viable intracellular bacteria at 4h, intracellular bacteria increased 16% at 24h, suggesting that Staphylococcus aureus is capable of proliferating in HaCaT ceils during that period. However the viable intracellular bacteria decreased 95% from 24h to72h. The addition of Forskolin （SUM） or PMA （SnM） led to 70% and 46% decrease of viable intracellular bacteria respectively compared with control at 24h. Conclusion： The clearance of intracellular bacteria by epidermal keratinocytes might be a host defense mechanism and the enhanced bacterial clearance by Forskolin or PMA Suggested that NADPH oxidases is involved in keratinocyte antibacterial activity.