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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。
Objective To investigate the effect of Cpn0425 recombinant protein on the induction of apoptosis and proinflammatory cytokines IL-8 and IL-1β in human monocytes in vitro and to provide experimental basis for further exploration of the molecular mechanism of Cpn infection. Methods The Cpn0425 protein coding gene of Chlamydia pneumoniae was amplified by PCR. The recombinant plasmid pGEX6p-2 / Cpn0425 was constructed and expressed in E. coli BL21. After being lysed by ultrasound, glutathione S-transferase (GST) The purified recombinant protein was purified by ToxinEraser column, and the THP-1 cells were stimulated with different concentrations of GST-Cpn0425 after removing endotoxin. The levels of IL-8 and IL-1β produced by stimulated THP-1 cells were detected by ELISA. -1 method was used to detect the proliferation or inhibition of THP-1 cells treated with GST-Cpn0425; apoptosis was detected by Annexin V-FITC-PI staining. Results GST-Cpn0425 could induce the expression of IL-8 and IL-1β in THP-1 cells. When the concentration of GST-Cpn0425 was increased to 6μg / ml (l8μg / ml), the amounts of IL-8 and IL- 716.11 ± 41.26) pg / ml and (32.91 ± 5.49) pg / ml. When 6μg / ml GST-Cpn0425 stimulated the cells for 6h, IL-8 and IL-1β were detected from the medium, while the peak value reached the peak at 24h. GST-Cpn0425 inhibited the proliferation of THP-1 cells in a dose-dependent manner. After THP-1 cells were treated with GST-Cpn0425 for 24 hours, the apoptosis of THP-1 cells was induced by GST-Cpn0425 with the highest apoptosis rate of (17.76 ± 4.2)%. Conclusion Cpn0425 protein can induce THP-1 cells to express and secrete pro-inflammatory cytokines IL-8 and IL-1β, which can inhibit the proliferation of THP-1 cells and induce apoptosis of THP-1 cells, which may be an important causative agent.