肺炎支原体感染RAW264.7细胞NLRP3炎性体及下游分子的表达

Expression of the NLRP3 inflammasome and its downstream molecules in RAW264.7 cells infected with Mycoplasma pneumoniae

目的观察肺炎支原体(Mycoplasma pneunoniae,Mp)感染RAW264.7细胞早期NLRP3炎性体及前炎性细胞因子的表达。方法将RAW264.7细胞随机分为5组,正常组用常规方法培养,不感染Mp;4个实验组RAW264.7细胞均用Mp感染4h,感染复数(细胞︰Mp)分别为1︰20、1︰40、1︰80、1︰100,采用FQ-PCR法检测细胞NLRP3、ASC、caspase-1mRNA表达和IL-1β、IL-18水平。结果 Mp感染复数1︰20、1︰40、1︰80、1︰100组NLRP3、ASC、caspase-1mRNA表达量分别为2.10±0.62、2.14±0.66、2.66±0.69、3.29±0.64和3.91±0.83、4.21±0.95、4.25±0.86、4.30±0.99和1.65±0.48、1.65±0.36、1.94±0.51、2.00±0.57,与正常对照组0.98±0.08、1.00±0.08、0.99±0.09比较,差异均有统计学意义(P<0.01);感染复数1︰100组IL-1β为200.00±9.25pg/ml,与对照组159.92±5.89pg/ml比较差异有统计学意义(P<0.01)。结论 Mp感染可诱导RAW264.7细胞NLRP3炎性体活化。NLRP3炎性体可能参与了Mp的早期感染。

Objective To observe the expression of the NLRP3 inflammasome and pro-inflammatory cytokines in RAW264.7 cells infected with Mycoplasrna pneumoniae. Methods RAW 264.7 cells were randomly divided into five groups. RAW264.7 cells in a normal group were not infected with M. pneumoniae. The remaining 4 groups of RAW264. 7 cells were infected with M. pneumoniae at a multiplicity of infection （MOI） of 1 ： 20, 1 ： 40, 1 ： 80, or 1 ： 100 for 4 hours. The expression of NLRP3, ASC, and caspase 1 mRNA was detected using FQ-PCR. Levels of 1L-1 13 and 1L-18 in RAW264.7 ceils were detected using ELISA. Results Expression of NLRP3 was 2.10 ± 0.62 for the group of cells infected with M. pneumoniae at an MOI of 1 ： 20, 2.14 ± 0.66 for the group of cells infected with A4. pneumoniae at an MOIof 1 ： 40, 2.66 ± 0.69 for the group of cells infected withM, pneurnoniaeat anMOIof l ： 80, and 3.29 ± 0. 64 for the group of cells infected with M. pneumoniae at an MOI of 1 ： 100 in comparison to 0.98 ± 0.08 for the nor real group. Expression of ASC was 3.91 ± 0.83 for the group of cells infected with M. pneumoniae at an MOI of 1 ： 20, 4.21 ±0.95 for the group of cells infected with M. pneumoniae at an MOI of 1 ： 40, 4.25 ± 0.86 for the group of cells infected with M. pneumoniae at an MOI of 1 ： 80, and 4.30 ± 0.99 for the group of cells infected with M. pneumoniae at an MOI of 1 ： 100 in comparison to 1.00± 0.08 for the nornml group. Expression of caspase-1 mRNA was 1. 65 ± 0.48 for the group of cells infected with M. pneumoniae at an MOI of 1 ： 20, 1.65 ± 0.36 for the group of ceils infected with M. pneurnoniae at an MOI of 1 ： 40, 1.94 ± 0.51 for the group of cells infected with M. pneumoniae at an MOI of 1 ： 80, and 2.00 ± 0.57 for the group of cells infected withM, pneurnoniae at an MOI of 1 ： 100 in comparison to 0.99 ± 0.09 for the normal group. Differences in the levels of expression were all statistically significant （P〈 0.01）. Levels of IL-1β （200 ± 9.25） pg/ml were significantly （P〈0.01） higher in cells infected with M. pneumoniae at an MOI of 1 ： 100 in comparison to those in the normal group （159.92 ± 5.89） pg/ml. Conclusion M. pneunmniae induces the activation of the NLRP3 inflammasome in RAW264.7 cells and the NLRP3 inflammasome may be involved in early infection with M. pneumoniae.