金黄色葡萄球菌生物膜对人离体鼻窦黏膜溶菌酶、分泌性白细胞蛋白酶抑制剂和糖蛋白340表达的影响

Staphylococcus aureus biofilm influences the expression of lysozyme,SLPI and gp340 in a human sinonasal explant model

目的:探讨不同表型金黄色葡萄球菌对人离体鼻窦黏膜组织溶菌酶、分泌性白细胞蛋白酶抑制剂(SLPI)和糖蛋白340(gp340)表达的影响。方法:采集10例经鼻内镜脑脊液鼻漏修补术患者的筛窦黏膜进行离体组织培养并建立金黄色葡萄球菌游离菌和生物膜感染模型。以激光共聚显微镜确认模型建立成功,分别在建模后8、16及24h以ELISA法测定溶菌酶、SLPI和gp340的分泌水平;24h后以RT-PCR、免疫组织化学和Western blot法检测培养组织中溶菌酶、SLPI和gp340的mRNA和蛋白表达水平。结果:溶菌酶、SLPI和gp340的分泌呈现出时间依赖性趋势,在接种8h和16h后,生物膜组溶菌酶、SLPI和gp340的分泌水平均较游离菌组和对照组显著增高(均P<0.05)。在mRNA水平,游离菌组上述分子的mRNA表达水平较对照组显著增高,生物膜组较游离菌组和对照组显著增高(均P<0.05);在蛋白水平,除Western blot法显示游离菌组和对照组溶菌酶表达无统计学差异外(P>0.05),游离菌组上述蛋白的表达均较对照组显著增高,生物膜组较游离菌组和对照组显著增高(P<0.05)。结论:金黄色葡萄球菌生物膜较游离菌更能诱导溶菌酶,SLPI和gp340在鼻窦黏膜组织中高表达,是影响宿主固有免疫的重要因素。

Objective：To investigate the influences of staphylococcus aureus in planktonic and biofilm forms on the expression of lysozyme,SLPI and gp340 in the human sinonasal explant model.Method：Mucosa samples from ethmoid sinus were collected from ten patients of cerebrospinal fluid leak and were cultured with and without S.aureus biofilms and planktonic cells.After the infection,the explant model was confirmed by CLSM,and the secretion of lysozyme,SLPI and gp340 was detected by enzyme-linked immunosorbent assay（ELISA）at 8,16,and24 hafter S.aureus challenge.Expressions of lysozyme,SLPI and gp340 in mRNA and protein levels after 24 hS.aureus challenge were detected using RT-PCR,immunohistochemistry and Western bolt assay respectively.Result：The secretion of lysozyme,SLPI and gp340 in the explant model was observed with a trend to increase in a timedependent manner.At 8and 16 hafter S.aureus challenge,the secretion of lysozyme,SLPI and gp340 in biofilms group was significantly higher than these in planktonic cells group and control group（P〈0.05）.S.aureus biofilms enhanced the mRNA expressions of lysozyme,SLPI and gp340 significantly compared with planktonic cells and controls,and the mRNA expressions in the explant model challenged by planktonic cells were significantly higher than controls（P〈0.05）.Although the Western bolt assay showed no differences between the lysozyme expression in the planktonic cells group and control group（P〉0.05）,the biofilms enhanced the expressions of lysozyme,SLPI and gp340 significantly compared with planktonic cells and controls（P〈0.05）.Conclusion：S.aureus biofilm induced the expressions of lysozyme,SLPI and gp340 to a higher level than planktonic cells,indicating that S.aureus biofilm was an influencing factor on the innate immune system.