肺炎链球菌感染小鼠早期肺组织细胞因子变化研究

The dynamic change of cytokines in lung tissues of mice in the early phase of Streptococcus pneumoniae infection

目的建立非致死性肺炎链球菌肺炎小鼠模型,探讨感染后72 h内肺组织细胞因子变化在清除肺炎链球菌感染中的作用。方法用灭菌PBS稀释处于对数生长期的肺炎链球菌标准株D39,对SPF级雌性Balb/c鼠（6～8周,15～18 g）滴鼻,收集Balb/c鼠支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）进行菌落计数、细胞分类计数,取肺组织进行菌落计数、病理学检查及细胞因子检测。结果感染2 h后BALF中细菌数量逐渐下降,72 h基本清除。肺匀浆细菌量于感染后6 h达高峰,6～24 h明显下降,72 h大部分细菌已清除。BALF中中性粒细胞百分比在感染后2 h开始增加,12 h达高峰,48 h降至正常水平;淋巴细胞百分比在感染后2 h开始增加,6 h达高峰,24 h逐渐降至正常水平。肺组织在感染后6 h出现炎症细胞浸润,48 h达高峰,随后炎症逐渐减轻,肺组织结构开始恢复。肺组织匀浆中的促炎因子IFN-γ、TNF-α、IL-6、IL-1及IL-17于感染后2 h明显增高（P〈0.05）,其中IFN-γ、TNF-α、IL-6及IL-1在6 h后逐渐降至正常,IL-17一直处于较高水平,感染12 h后仍高于正常水平（P〈0.05）,之后逐渐降至正常;抗炎因子TGF-β与IL-10于感染后2 h明显增高（P〈0.05）,其中TGF-β于感染后6 h达高峰后逐渐降至正常水平,而IL-10一直持续在较高水平直至感染后48 h才开始下降（P〈0.05）。结论肺组织感染后早期细胞因子变化除在固有免疫中发挥重要作用外,可能在启动适应性免疫应答和免疫调节中也有重要作用,促炎及抗炎因子的动态平衡在清除细菌的同时也避免了过强的炎症反应所导致的组织损伤。

The study was designed to establish nonfatal Streptococcus pneumoniae mouse model and investigate the role of the cytokines in clearing Streptococcus pneumoniae within 72 hours after infection. Adult SPFBalb/c mice （female, 6-8 week, 15-18 g） were infected with 50 txl Streptococcus pneumoniae D39 by nose dropping at the concentration of 3×10^6 cfu/ml. Then BALF of experimental mice were collected for bacterial counting andcells counting; the lung tissues were collected for bacterial counting, histopathological examination and cytokine detection. We found the bacterial population of the BALF dropped gradually at 2 h after inoculation, then droppedto normal level at 72 h after inoculation. The bacterial population of the lung homogenate peaked at 6 h after inoculation, and dropped obviously from 6 h to 24 h, and mostly cleared in 72 h after inoculation. The percentage oflung neutrophils increased obviously at 2 h after inoculation, and peaked at 12 h, then dropped to normal level at 48 h. The percentage of lymphocytes in lung homogenate increased obviously at 2 h after inoculation, and peaked at6 h, then dropped to normal level at 24 h. The inflammation cell infiltration in lung tissue was found at 6 h after inoeulation, peaked at 48 h, and then lung tissue began to recover. The levels of pro-inflammation eytokines in lunghomogenate such as IFN-γ、TNF-α、IL-6、IL-1 and IL-17 were significantly higher （P 〈 0.05） at 2 h afterinoculation, then the levels of IFN-γ、TNF-α、IL-6 and IL-1 dropped gradually to normal after 6 h, whilethe level of IL-17 kept high level in the first 12 h after inoculation （P 〈 0.05） then dropped to normal level. Inconclusion, the changes of the cytokines in lung tissuesof early phase of infection may play an important role not only in the innate immune response but also in initiating adaptive immune response. The dynamic balance of cytokines could promote the recruitment of neutrophils andlymphocytes to clear the bacteria, at the same time avoid lung tissue damage caused by the excessive inflammatory response.