无菌大鼠深二度烫伤感染金黄色葡萄球菌机体炎性反应的研究

Study on Inflammatory Response of the Staphylococcus aureus Infected Deep Second Degree Scald Germ-free Rats

目的为探究无菌大鼠深二度烫伤创面感染金黄色葡萄球菌后机体的炎性反应,试验应用无菌大鼠及SPF级大鼠分别建立深二度烫伤模型和烫伤后金黄色葡萄球菌感染模型,分析愈合过程、炎性反应及病理变化。方法20只6周龄雌性Wistar无菌大鼠,在超净工作台内采用恒温恒压烫伤仪94℃作用8 s造成大鼠深二度烫伤,随机分为两组,一组伤口感染浓度为1×108CFU/m L金黄色葡萄球菌0.5 m L,一组不做处理,创面保持无菌状态;20只6周龄雌性SPF级Wistar大鼠,在相同条件下烫伤,随机分为两组,一组伤口感染浓度为1×10~8CFU/m L金黄色葡萄球菌0.5 m L,一组不做处理。分别观察4组大鼠的伤口结痂时间、脱痂时间和愈合时间,并在烫伤前(0 h),烫伤后24 h、48 h、3d、5 d、7d、10 d、14 d时取血清,检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1α(IL-1α)和表皮生长因子受体(EGFR)的变化;烫伤后24 h、3d、7 d、10 d伤口处皮肤HE染色,观察病理变化。结果感染金黄色葡萄球菌无菌大鼠伤口脱痂及愈合时间显著短于其他3组(P<0.05);感染金黄色葡萄球菌SPF级大鼠,结痂、脱痂及愈合时间显著长于其他3组(P<0.05);感染金黄色葡萄球菌SPF级烫伤大鼠组由于双重损伤使血清炎性因子TNF-α、IL-1α水平升高,且在烫伤后72 h显著高于其他3组(P<0.05);无菌大鼠由于对烫伤及金黄色葡萄球菌刺激反应激烈,烫伤后血清TNF-α、IL-1α和EGFR水平迅速升高,且在烫伤24 h时显著高于2组SPF大鼠(P<0.05),在烫伤48 h时血清EGFR水平显高于两组SPF大鼠(P<0.05)。病理显示,感染金黄色葡萄球菌SPF烫伤大鼠伤口处新生血管及肉芽组织生成时间均长于其他3组;感染金黄色葡萄球菌无菌烫伤大鼠伤口处新生血管及肉芽组织生成时间均短于其他3组。结论无菌大鼠由于其免疫系统处于休眠状态,受到烫伤和金黄色葡萄球菌的双重刺激,迅速激活其全身免疫系统,加快了伤口愈合;SPF烫伤大鼠感染金黄色葡萄球菌后产生的多种免疫逃避分子加重了机体的炎性反应,延缓了伤口愈合。

Objective We established a deep second-degree scald injury model which was infected with Staphylococcus aureus with germ-free rats and SPF rats,then we analyze the healing process,inflammatory response,and pathological changes about the four groups.Method Deep second degree scald wound model was performed in 20 six weeks old female germ-free rats at a temperature of 94℃,8 s in a clean bench.The scald germfree rats were randomly divided into two groups.One group was infected with Staphylococcus aureus with a concentration of 1×10~8 CFU/m L in the wound;Another group was not infected.20 six weeks old female SPF Wistar rats were performed second degree scald wounds as the germ-free rats.The scab generating time,dislocation time and healing time of the four groups were observed,and serum samples were taken to detect the changes of IL-1α,TNF-αand EGFR before burned(0 h)and 24 h,48 h,3 d,5 d,7 d,10 d,14 d after burned.The skins were taken to observe the pathological changes at 24 h,3 d,7 d and 10 d after burned.Result The Staphylococcus aureus stained germ-free rats had a short period of decrustation and healing time(P<0.05),while the Staphylococcus aureus stained SPF rats had a long period of scab generating time,decrustation and healing time(P<0.05).Because of the double damage,the level of TNF-αand IL-1αwere elevated and they were significantly higher than the other three groups after 72 h scald(P<0.05);The germ-free rats response stimulus to scald and Staphylococcus aureus,so the level of TNF-α、IL-1αand EGFR were elevated and they were significantly higher the SPF rats at 24 h after burned.The level of EGFR about the Staphylococcus aureus stained germ-free rats was significantly higher than SPF rats at 48 h after burned.The pathological showed time of new blood vessels and granulation tissue formation about the Staphylococcus aureus stained SPF rats were significantly longer than the other three groups and time of new blood vessels and granulation tissue formation about the Staphylococcus aureus stained germ-free rats were significantly shorter than the other three groups.Conclusion The immune system of germ-free rats was in a state of dormancy,when it was stimulated by scald and Staphylococcus aureus,the systemic immune was activated and the wound healing was accelerated;the variety of immune evasion molecules secreted by Staphylococcus aureus aggravated the inflammatory response and wound healing was delayed.