人β防御素3体内抑制耐甲氧西林葡萄球菌内植物生物膜感染的机制研究

The mechanism of human-defensin 3 in methicillin β-resistant Staphylococcus aureus-induced infection of implant biofilm in the rat tibial bone marrow

目的明确耐甲氧西林金黄色葡萄球菌(MRSA)注入大鼠胫骨的骨髓腔后,应用人β防御素3(HBD-3)抑制耐药菌植入物表面生物膜感染的机制。方法将MRSA注入SD大鼠左胫骨骨髓腔中构建耐药植入物细菌生物膜感染的模型:各实验组(包括空白对照组,HBD-3组和万古霉素组,每组20只)髓腔感染24小时后腹膜内注射药物,对照组注射2 mL生理盐水,HBD-3组注射2 m L 8 g/mL(1 MIC)HBD-3,万古霉素组注射2 mL0.5 g/mL(1 MIC)万古霉素,每组分别在腹腔注射后第1,7,14和21天处死5只;激光共聚焦显微镜观察植入物表面上的生物膜和活菌的数量,免疫组化评估NF-κB和TLR-4的表达,ELISA法检测IL-10及TNF-。结果对照组白细胞数和中性粒细胞的百分比,以及生物膜形态和对照组的存活细菌的数目随时间逐渐增加,而HBD-3组和万古霉素组随时间下降(<0.05)。HBD-3组NF-κB和TLR-4表达量在第1天增加,在第7天达到峰值,在第14天开始下降,每个时间点的相对差异均有统计学差异(<0.05)。NF-B和TLR-4表达量各时间点在HBD-3组中较对照组和万古霉素组中均有统计学差异。IL-10及TNF-4的表达在对照组中均显著高于其它两组(<0.05)。结论β防御素3可以通过调节炎症反应和免疫应答,抑制大鼠胫骨骨髓MRSA引起的耐药菌植入物生物膜感染。

Objective To study the mechanism of human β-defensin 3 （HBD-3） in methicillin-resistant Staphylococcus aureus （MRSA）-induced infection of implant drug-resistant bacteria biofilm in the rat tibial bone marrow. Methods The SD rats were selected to construct the model of MRSA-induced implant biofilm infection in the left tibial bone marrow. The drugs were intraperitoneally injected after 24 h medullary cavity infection, and the experimental groups included the model group, HBD-3 group, and vancomycin group （20 rats in each group）. The model group was injected with 2 mL saline, HBD-3 group was injected with 2 mL 8 μg/ml （1MIC） HBD-3 and vancomycin group was injected with 2 mL 0.5μg/mL （1 MIC） vancomycin. Five animals that had been injected in each group were sacrificed on the 1st, 7th, 14th, and 21st day, respectively. The laser scanning confocal microscopy was used to observe the morphology of the biofilm and the number of viable bacteria. Immunohistochemical staining was adopted to test the expressions of NF-κB and TLR-4, and ELISA was used to test IL-10 and TNF-α expression levels. Results The percentage of the neutrophile granulocytes, total white blood cells and the number of viable bacteria in the model group was gradually increased, while those in the HBD-3 and vancomycin groups were decreased gradually （P〈 0.05）. The NF-κB and TLR-4 expressions in the HBD-3 group began to increase on the 1st day, reached the peak on the 7th day and began to be fallen on the 14th day; those in the HBD-3 group were significantly higher than the model group and vancomycin group at each time point （P〈 0.05）. The IL-10 and TNF-α expressions in the model group at each time were significantly higher than the other two groups （P〈0.05）. Conclusion HBD-3 can inhibit the bacterial growth by regulating inflammations and immune responses in the MRSA-induced implant biofilm infection in the rat tibial bone marrow.