肺炎嗜衣原体CPAF重组蛋白致小鼠肺组织炎症及对炎症细胞因子的影响

Study on the inflammatory injury of lung tissue and influence on inflammatory cytokines in mouse induced by recombinant protein CPAF from Chlamydophila pneumoniae

目的：研究肺炎嗜衣原体（Chlamydophila pneumoniae,Cpn）蛋白酶样活性因子（CPAF）的181400aa基因（CPAFm）的重组蛋白在BALB/c小鼠体内引起的肺部炎症改变及对炎症细胞因子TNF-α和IL-6的水平的影响,为进一步探索CPAF在Cpn感染中的致病作用提供实验依据。方法：将纯化的CPAFm重组蛋白于鼻内或尾静脉注射BALB/c小鼠,HE染色观察肺组织病理形态学改变,计小鼠外周血与支气管肺泡灌洗液（Bronchoalveolar lavage fluids,BALF）中白细胞总数,ELISA方法检测血清和BALF中的TNFα-和IL-6的水平。结果：实验组BALB/c小鼠的肺组织出现中性粒细胞、淋巴细胞等炎症细胞浸润,对照组与正常组BALB/c小鼠肺组织未见明显病理改变。鼻内滴入重组蛋白实验组BALB/c小鼠BALF中白细胞总数明显高于GST对照组（P〈0.05）、PBS对照组（P〈0.05）和正常组（P〈0.01）;鼻内滴入和尾静脉注射重组蛋白实验组BALF中炎症因子IL-6、TNF-α的水平明显高于GST、PBS对照组与正常组（均P〈0.01）;鼻内滴入重组蛋白实验组外周血中炎症因子IL-6和TNF-α水平显著高于GST、PBS对照组和正常组（P〈0.05）;尾静脉注射重组蛋白实验组外周血中炎症因子IL-6的水平明显高于相应的对照组（均P〈0.01）。结论：CPAFm重组蛋白有致病性,能引起BALB/c小鼠肺组织的炎症损伤和炎症因子TNF-α、IL-6的水平的升高,肺损伤与BALB/c小鼠体内炎症细胞增多和炎症因子TNF-α、IL-6的水平的升高相关。

Objective：To provide experimental basis for exploring pathogenic mechanism of Chlamydophila pneumoniae,and to investigate the changes of pulmonary inflammation and inflammatory cytokines including TNF-α and IL-6 levels in mouse model.Methods：The gene fragment of Chlamydial protease-like activity factor（CPAF）from C.pneumoniae（CPAFm） was cloned,which encoded 181～400aa of the protein.The gene fragment was inserted into plasmid（name） and expressed in Escherichia coli.The purified recombinant GST-CPAFm protein was instilled into anterior nose or injected into the caudal vein of BALB/c mice 3 times on the day 1,3 and 6.On the day 7,the mice were killed.Pathological alteration was observed by hematoxylin and eosin staining.The total white blood cells of peripheral blood and bronchoalveolar lavage fluid（BALF） was counted with blood cell counting plate,and the concentration of TNF-α and IL-6 in both plasma and BALF was detected by Enzyme-linked immunoadsordent assay technique.Results：（1） Histological examination showed a marked inflammatory immune responses in the mouse lung tissues after intranasal application or intravenous injection of 100 μg GST-CPAFm and there were obvious leukocyte infiltration,such as neutrophils,lymphocytes and macrophages in bronchiole and perivascular.There were no obvious pathological changes in the control and normal group BALB/c mice.（2） The percentage of peripheral blood neutrophils in GST-CPAFm intravenously group was significantly higher than that of the GST group（P〈0.05）,PBS group（P〈0.05） and normal group（P〈0.01）.（3） The level of inflammatory cytokines IL-6 of the peripheral blood in GST-CPAFm intranasally group was significantly higher than that of the GST group（P〈0.01）,PBS group（P〈0.01） and normal group（P〈0.01）,and the level of inflammatory cytokines TNF-α were significantly higher than in the GST group（P〈0.05）,PBS group（P〈0.01） and normal group（P〈0.01）;and the level of inflammatory cytokines IL-6 in GST-CPAFm intravenously group was significantly higher than that of the GST group （P〈0.01）,PBS group（P〈0.01） and normal group P〈0.01）.（4） The level of inflammatory cytokines IL-6 and TNF-α of BALF in GST-CPAFm intranasally group were significantly higher than that of the GST,PBS and normal group（P〈0.01）;and the level of inflammatory cytokines IL-6 and TNF-α of BALF in GST-CPAFm intravenously group were significantly higher than the GST,PBS and normal group（P〈0.01）.Conclusion：Therecombinant GST-CPAFm protein has pathogenicity and could induce obvious pulmonary inflammatory reaction and lung injury,which are relevant to the increase of inflammatory cells and the expression of inflammatory cytokines including TNF-α and IL-6 in BALF.