肺炎链球菌荚膜多糖加重C57BL/6小鼠中耳炎的炎症应答和中耳组织损伤

Streptococcus pneumoniae capsular polysaccharide aggravate inflammation and tissue injury in acute otitis media of C57BL/6 mice

摘要:

目的：研究肺炎链球菌（Streptococcus pneumoniae，S.pn）荚膜多糖（capsular polysaccharide，CPS）对C57BL/6小鼠急性中耳炎的炎症应答和组织损伤的作用。方法：首先通过同源重组的方式构建TIGR4Δcps菌株，然后经鼓膜穿刺，分别接种5 μl/耳的TIGR4和TIGR4Δcps于C57BL/6小鼠的双侧中耳腔，对照组采用相同方法接种等体积无菌磷酸盐缓冲液（phosphate buffered saline，PBS）。观察小鼠发病情况，分别于感染后1、3、5 d制备中耳组织切片，HE染色观察中耳组织损伤及炎性细胞浸润变化，同时收集中耳灌洗液（middle ear lavage fluids，MELF），检测MELF中细菌载量、炎性细胞数量和细胞因子含量。结果：成功构建了TIGR4Δcps菌株。与TIGR4组相比，TIGR4Δcps组小鼠中耳上皮的损伤更轻，中性粒细胞募集减少（第1天时P=0.004；第3天时P=0.000；第5天时P=0.000），MELF中细胞因子TNF-α和IL-6水平更低（第1天时P=0.076、P=0.000；第3天时P=0.002、P=0.003；第5天时P=0.000、P=0.000），但细菌清除更快（第1天时t=2.659，P=0.029；第3天时t=3.717，P=0.006）。结论：在S.pn TIGR4致小鼠急性中耳炎过程中，CPS具有加重炎症应答和组织损伤，并导致宿主对S.pn的清除减慢的作用。

Abstract:

Objective：To study the effects of Streptococcus pneumoniae（S.pn） capsular polysaccharide（CPS） on acute otitis media of C57BL/6 mice. Methods：TIGR4Δcps strain was built by homologous replacement construction. Wild TIGR4（5 ?滋l/ear） or TIGR4Δcps was respectively inoculated into bilateral ear cavity by puncturing the tympanic membrane；an equal amount of sterile phosphate buffered saline（PBS） was injected into the control group. The status of mice was detected every day；inflammation of middle ear ep－ithelia that deal with wild TIGR4 or TIGR4Δcps was observed through HE staining on 1，3，5 d after inoculation. The role of CPS in cell recruitment and bacterial clearance was observed by cell counts and clonal formation unit counts，and the inflammatory cytokines in the middle ear lavage fluid（MELF） were measured to compare the level of inflammation. Results：TIGR4Δcps mutant strain was con－structed successfully. Compared with those of TIGR4 group，mice inoculated with TIGR4Δcps showed lighter damage of middle ear epithelial，lower level of TNF-α and IL-6 in MELF（1 d：P=0.076，P=0.000；3 d：P=0.002，P=0.003；5 d：P=0.000，P=0.000），fewer recruited neutrophils（1 d：P=0.004；3 d：P=0.000；5 d：P=0.000），and faster bacterial clearance（1 d：t=2.659，P=0.029；3 d：t=3.717，P=0.006）. Conclusion：In the mice model of S.pn induced acute otitis media，CPS exacerbates inflammation and tissue injury，and delays clearance of bacteria.