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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 in Streptococcus pneumonia (S.pn) on the innate immune response in mice with pneumonia. Methods BALB / c mice were randomly divided into blank control group, D39 infection group and △ dnaJ infection group, respectively, by intranasal instillation of 30μl sterile PBS, 30μl containing 2 × 107cfu S.pn D39 and △ dnaJ (dnaJ gene defect Strain) bacteria, replicating mouse pneumonia model. The mice were sacrificed at 6, 12, 24, 36 and 48 h after infection. The lungs were harvested and the supernatant was separated. The expression of TNF-α, IL-1β, IL- HE staining was used to observe the changes of inflammatory reaction in the lungs of mice infected 12 h after infection. The murine macrophage cell line RAW264.7 was infected with S.pn D39 and △ dnaJ in vitro, the ratio of bacteria to cells was 100: 1, After 3 h, the cell culture supernatant was separated and the expression of TNF-α and IL-6 was detected by ELISA. Real-time PCR and Western blot were used to detect the mRNA transcription of pattern recognition receptor TLRs and the phosphorylation level of inflammation-related signal p38MAPK in lung tissue of mice at 12 h after infection. Results Compared with D39 infection group, the intensity of inflammatory reaction in lung tissue of △ dnaJ infection group was decreased, and the levels of TNF-α, IL-6 and IL-1β and other proinflammatory cytokines peaked and peaked, The levels of TNF-α and IL-6 secreted at 3 h after infection decreased significantly (P <0.01 or P <0.05). The mRNA transcription levels of Tlr2 and Tlr13 in lung tissue were significantly decreased (P <0.05), and the phosphorylation of p38 MAPK also significantly decreased . Conclusions Defective S. pneumoniae dnaJ gene can down-regulate the inflammatory response, proinflammatory cytokine secretion and activation of intracellular signaling molecules in mice with pneumonia infection. It may also affect the expression of Trl2 and Tlr13 mRNA in lung tissue of mice after infection, The study of the body of Streptococcus pneumoniae DnaJ protein laid the foundation for the natural immune response molecular mechanism.