肺炎链球菌dnaJ基因缺陷对小鼠肺炎感染模型天然免疫应答的影响

Effect of dnaJ gene defect on natural immune response in mice infected with Streptococcus pneumoniae

目的探讨肺炎链球菌(Streptococcus pneumonia,S.pn)dnaJ基因缺陷对肺炎感染模型小鼠天然免疫应答的影响。方法将BALB/c小鼠随机分为空白对照组、D39感染组和△dnaJ感染组,分别经鼻腔滴注30μl无菌PBS、30μl含2×107cfu的S.pn D39和△dnaJ(dnaJ基因缺陷株)菌液,复制小鼠肺炎感染模型。于感染后6、12、24、36和48 h处死小鼠,取肺组织,匀浆后分离上清,ELISA检测促炎因子TNF-α、IL-1β、IL-6和IFNγ的表达水平,HE染色观察感染12 h的小鼠肺组织炎症反应变化;将小鼠巨噬细胞株RAW264.7体外感染S.pn D39和△dnaJ,细菌与细胞的比例为100∶1,感染1、2、3 h后分离细胞培养上清液,ELISA检测TNF-α和IL-6的表达水平。实时定量PCR和Western blot分别检测感染12 h的小鼠肺组织中模式识别受体TLRs基因mRNA的转录水平和炎症相关信号p38MAPK的磷酸化水平。结果与D39感染组相比,△dnaJ感染组小鼠肺组织炎症反应强度下降,TNF-α、IL-6和IL-1β等促炎因子水平达峰时间延迟,且峰值降低,RAW264.7细胞感染3 h分泌TNF-α和IL-6显著减少(P<0.01或P<0.05);小鼠肺组织Tlr2和Tlr13基因mRNA转录水平显著降低(P<0.05),p38MAPK的磷酸化水平也明显下降。结论肺炎链球菌dnaJ基因缺陷可下调肺炎感染模型小鼠的炎症反应、促炎因子分泌及胞内信号分子的激活,也可影响小鼠感染后肺组织中Trl2和Tlr13基因mRNA的表达,为进一步研究机体对肺炎链球菌DnaJ蛋白的天然免疫应答分子机制奠定了基础。

Objective To investigate the effect of dnaJ gene defect on natural immune response in mice infected with Streptococcus pneumonia（S, pn）. Methods BALB / c mice were randomly divided into blank control, D39 infection and AdnaJgroups, and infected with 30 μl of sterile PBS,30 Ixl （2 × 107cfu） of D39 strain and 30 μl （2 × 107 cfu） of AdnaJ strain （dnaJ gene defective strain） of S. pn by intranasal drip respectively to copy mouse model of S. pn infection. Four mice were killed 6, 12, 24, 36 and 48 h after infection respectively, of which the lung tissues were collected and homogenized. The supernatants were collected and determined for the expression levels of TNF-α, IL-1β, IL-6.and IFNγ by ELISA. The inflammatory reactions in lung tissues of mice 12 h after infection were observed by HE staining. S. pn D39 and AdnaJ strains were infected to mouse macrophage RAW264. 7 strain in vitro, each at a ratio of 100 ： 1. The culture supernatant of RAW264. 7 strain was collected 1,2 and 3 h after infection and determined for expression levels of TNF-c~ and IL-6 by ELISA. The transcription levels of mRNAs of TLRs in mouse lung tissue 12 h after infection were determined by real-time quantitative PCR, while the phosphorylation level of p38MAPK by Western blot. Results As compared with those in D39 infection group, the lung inflammatory response of mice in AdnaJ infection group was down-regulated, the appearance of secretion peaks of TNF-α, IL-α and IL-1β were delayed, and the peak values decreased, while the secretion levels of TNF-α and IL-6 3 h after infection with RAW264. 7 cells decreased significantly（P 〈 0. 01 or P 〈 0. 05）;meanwhile,the transcription levels of Tlr2 and Tlrl3 mRNAs（ P 〈 0. 05） as well as the phosphorylation level of p38MAPK decreased significantly. Conclusion The dnaJ gene defect of S. pn down-regulated the inflammatory response, secretion of proinflammatory cytokines and activation of eelluar signal molecules, and affected the expressions of Tit2 and Tlrl3 mRNAs in lungs of mice infected with S. pn, which laid a foundation of further study on the molecular mechanism of natural immune response to DnaJ protein of S. pn.