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目的:以沙眼衣原体野生株(wild type,WT)及无质粒株(plasmid free,PF)感染新生鼠诱导肺炎,探究衣原体质粒对菌株致病力的影响。方法:将出生24 h内的48只BALB/c新生鼠随机分成4组,每组12只,WT组感染有质粒的沙眼衣原体血清型D野生菌株,PF组感染同型无质粒株,磷酸盐缓冲液(phosphate buffer,PBS)组予相同剂量PBS处理,正常组无处理。于4、7、11 d各处死4只取标本,逆转录PCR检测肺组织内omp A基因m RNA水平;肺组织苏木精-伊红(H&E)染色及病理评分;髓过氧化物酶(MPO)免疫组化染色分析肺中性粒细胞浸润情况;酶联免疫吸附试验(ELISA)检测肿瘤坏死因子(tumor necrosis factor-α,TNF-α)及干扰素-γ(interferon-γ,IFN-γ)。报告基因分泌型胚胎碱性磷酸酶(SEAP)实验体外检测TLR2、NF-κB激活。结果:感染后肺组织衣原体培养阴性,而omp A基因m RNA检测及MPO免疫组化染色阳性,证实WT及PF菌株皆可感染新生鼠肺,引起间质性改变伴中性粒细胞浸润,PF组的肺组织病理评分(1.10±0.19)及PMN计数(21.06±10.92)均低于WT组分别为(1.44±0.17)和(43.90±17.17),P值分别为0.048和0.005;ELISA示WT组新生鼠肺内TNF-α(623.35±37.23)和IFN-γ(2206.83±854.22)水平较对照组分别为(470.93±20.83)和(737.97±79.89)升高(P值分别为0.000和0.038),而PF组分别为(531.59±34.14)和(1099.86±427.39)与对照组比较,无统计学差异(P值均>0.05);SEAP实验显示,PF株可激活TLR2信号(F=483.199,P=0.000),但程度较WT组有下降(P=0.000),激活NF-κB的能力明显受限(F=229.884,P=1.000),与对照组无统计学差异。结论:沙眼衣原体质粒缺乏使其感染肺部的致病力减弱,可能与衣原体质粒潜在的致病因子影响TLR2及NF-κB的活化有关。
OBJECTIVE: To infect virulent pneumonia induced by wild-type (WT) and plasmid-free (PF) strains of chlamydia trachomatis and explore the effect of the chlamydia plasmid on the virulence of the strain. Methods: 48 BALB / c neonatal mice within 24 hours of birth were randomly divided into 4 groups (n = 12). WT group was infected with plasmid of C. trachomatis serotype D wild strain, while PF group was infected with the same type of plasmid without plasmid. Phosphate buffered The rats in the phosphate buffered saline (PBS) group were treated with the same dose of PBS, while those in the normal group were untreated. At 4, 7 and 11 days, 4 samples were taken from each group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA level of omp A gene in lung tissue. H & E staining and pathological score of lung tissue were determined. Myeloperoxidase (MPO) immunohistochemistry was used to analyze the infiltration of neutrophils in the lung. The levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were measured by enzyme linked immunosorbent assay (ELISA) γ). The reporter gene secreted embryo alkaline phosphatase (SEAP) assay was used to detect TLR2 and NF-κB activation in vitro. Results: After infection, the chlamydial culture of lung tissue was negative. However, the omp A gene m RNA and MPO immunohistochemical staining showed that both WT and PF strains could infect newborn rat lung, causing interstitial changes with neutrophil infiltration, PF The lung histopathological score (1.10 ± 0.19) and PMN count (21.06 ± 10.92) were significantly lower in WT group than in WT group (1.44 ± 0.17 and 43.90 ± 17.17, P = 0.048 and 0.005, respectively) The levels of TNF-α (623.35 ± 37.23) and IFN-γ (2206.83 ± 854.22) in neonatal rats were (470.93 ± 20.83) and (737.97 ± 79.89) higher than those in the control group respectively (P = 0.000 and 0.038, respectively) However, there was no significant difference in PF group (531.59 ± 34.14) and (1099.86 ± 427.39) compared with the control group (all P> 0.05). The SEAP assay showed that the PF strain activated TLR2 signal (F = 483.199, 0.000). However, the level of NF-κB was significantly decreased (F = 229.884, P = 1.000), but there was no significant difference with the control group. Conclusion: The lack of plasmids of Chlamydia trachomatis weaken the pathogenicity of the infected Chlamydia trachomatis, which may be related to the potential virulence factors of Chlamydia pneumoniae affecting the activation of TLR2 and NF-κB.