【摘要】 目的研究沙眼衣原体(Chlamydia trachomatis,Ct)pORF5质粒蛋白诱导THP-1细胞产生IL-1β的分子机制,为阐明Ct致病机制提供实验依据。方法将原核表达重组体pGEX-6p/pORF5转化大肠埃希菌,表达并纯化GST-pORF5融合蛋白,融合蛋白经蛋白酶酶切后制备不含GST标签的pORF5蛋白;用不同浓度的pORF5蛋白体外刺激THP-1细胞,ELISA测定不同时间IL-1β水平;分别用NALP3siRNA、ASC siRNA、Caspase-1抑制剂和p38抑制剂预处理THP-1细胞,再用pORF5蛋白刺激THP-1细胞24h,ELISA测定IL-1β含量,Real-time PCR测定IL-1β和NALP3炎性体mRNA的表达,Western blot分析Caspase-1的表达及p38磷酸化水平。结果 pORF5蛋白以剂量和时间依赖的方式刺激THP-1细胞产生IL-1β,24μg/ml pORF5蛋白刺激24h时IL-1β的表达水平达到峰值(495.1±55.5pg/ml);pORF5蛋白能促进THP-1细胞中NALP3炎性体mRNA的表达;NALP3-siRNA、ASC-siRNA及Caspase-1抑制剂预处理THP-1细胞后,IL-1β分泌量分别降低37.7%、71.3%和40.1%;p38抑制剂能降低NALP3炎性体mRNA及IL-1β分泌量(P<0.01),但抑制NALP3炎性体后p38磷酸化水平未受影响(P>0.05)。结论 pORF5质粒蛋白通过激活p38MAPK和NALP3信号通路共同调控IL-1β的产生和分泌。更多还原

【Abstract】 Objective To investigate the molecular mechanism of 1 L-1βproduction induced by the pORF5 plasmid protein of Chlamydia trachomatis in THP-1 cells to lay the foundation for further study of the pathogenesis of C.trachomatis. Methods A pGEX-6 p/pORF5 prokaryotic expression vector was transformed into E.coli to express the fusion protein GST-pORF5.The fusion protein was digested with protease to yield pORF5 protein without the GST tag.pORF5 protein was used to stimulate THP-1 cells at different concentrations and different time points,and IL-1βwas detected with ELISA.After treatment with NALP3 siRNA,ASC siRNA,a caspase-1 inhibitor,or a p38 inhibitor,THP-1 cells were subsequently stimulated with pORF5(24μg/ml)for 24 h.Levels of IL-1βand NALP3 inflammasome mRNA were detected with ELISA or real-time PCR.Caspase-1 activity and p38 phosphorylation were determined using Western blotting. Results The pORF5 plasmid protein induced THP-1 cells to produce IL-1βin a dose-and time-dependent manner,and IL-1βproduction peaked at a concentration of 24μg/ml after 24 h.pORF5 protein increased expression of NALP3 inflammasome mRNA and the levels of IL-1βby 37.7% after treatment with NALP3 siRNA,by 71.3% after treatment with ASC siRNA,and by 40.1% after treatment with the caspase-1 inhibitor.The p38 inhibitor significantly(P<0.01)reduced pORF5-induced IL-1βproduction and expression of NALP3 inflammasome mRNA,but p38 phosphorylation did not differ significantly(P>0.05)after inhibition of NALP3 inflammasome activation. Conclusion pORF5 plasmid protein induced the production of IL-1βvia NALP3 and p38 MAPK signaling pathways.更多还原