Cpn0425重组蛋白诱导细胞凋亡和产生前炎症因子的研究

目的研究Cpn0425重组蛋白在体外诱导人单核细胞产生前炎症细胞因子IL-8和IL-1β的水平及诱导细胞凋亡的作用,为进一步探索Cpn感染致病的分子机制提供试验依据。方法 PCR扩增肺炎嗜衣原体Cpn0425蛋白编码基因,构建pGEX6p-2/Cpn0425重组质粒,在E.coli BL21中诱导表达,超声裂解后用谷胱甘肽S-转移酶（glutathione S-transferase,GST）纯化树脂纯化重组蛋白,ToxinEraser纯化柱去除内毒素后用不同浓度的GST-Cpn0425刺激THP-1细胞;ELISA法检测经刺激后的THP-1细胞产生的IL-8和IL-1β水平;以WST-1法检测经GST-Cpn0425处理后THP-1细胞的增生或抑制作用;用AnnexinV-FITC-PI染色法检测细胞凋亡情况。结果GST-Cpn0425能诱导THP-1细胞表达IL-8和IL-1β,当浓度增加到6μg/m（l8μg/ml）时,所产生的IL-8和IL-1β量最大,浓度分别为（716.11±41.26）pg/ml和（32.91±5.49）pg/ml。当6μg/ml GST-Cpn0425分别刺激细胞6h后即可从培养基中检测到IL-8和IL-1β,而刺激24h产生的量则达到高峰。GST-Cpn0425以剂量依赖方式抑制THP-1细胞增殖;GST-Cpn0425处理THP-1细胞24h后能诱导其发生凋亡,其细胞凋亡率最高为（17.76±4.2）%。结论Cpn0425蛋白能诱导THP-1细胞表达并分泌前炎症细胞因子IL-8和IL-1β;既能抑制THP-1细胞增殖,又能诱导其凋亡;因而可能是一个重要的致病因素。

Expression of human proinflammatory cytokines from monocytic cells induced by Cpn0425 recombinant protein from Chlamydophila pneumoniae and apoptosis

Objective To investigate the expression and production of proinflamatory cytokines including IL-8 and IL-1β in human monocytic cells（THP-1）;and apoptosis in human monocytic cells,to investigate the potential pathogenicity of Chlamydia and its molecular mechanisms responsible for the induction of proinflammatory cytokines and apoptosis.Methods The gene of Cpn0425 was amplified by PCR,which was used to construct the recombinant plasmid of pGEX6p-2/Cpn0425,induced expression in E.coli BL21,it was purified with glutathione S-transferase（GST） resin chromatography of Novagen;THP-1 cells were stimulated by different concentrations of the recombinant protein of Cpn0425 from Chlamydophila pneumoniae after sonication,after removal of endotoxin by purification columns of ToxinEraser;the expression of IL-8 and IL-1β were tested and proirferation by ELISA.Zhe inhibition of cell treated with Cpn0425 was assessed by WST-1.Cell apoptosis was detected by Annexin-V-FITC-PI.Results The recombinant protein of Cpn0425 stimulated THP-1 cell expressing the mRNA of IL-8 and IL-1β and produce proinflamatory cytokines including IL-8 and IL-1β in a dose and time-dependent manner;Cell proliferation of THP-1 was inhibited in a dose-dependent manner of GST-Cpn0425;The apoptosis was induced 24 hours after treatment of THP-1 cells with GST-Cpn0425,its highest apoptosis rate reached 17.76±4.2%.Conclusions The recombinant protein of Cpn0425 can induce the expression of THP-1 cells and secretion of proinflammatory cytokines IL-8 and IL-1β,inhibit the proliferation and apoptosis of THP-1 cells;therefore may be an important virulence factor.