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目的探讨重组嗜肺军团菌鞭毛蛋白A(rflaA)对RAW264.7巨噬细胞分泌白细胞介素6(IL-6)、IL-1β的影响及其机制。方法采用(0.000、0.125、0.250、0.500、1.000、2.000、4.000、8.000)μg/mL rflaA处理RAW264.7细胞,并设细胞对照组,用CCK-8法确定rflaA的半数效应剂量(EC_(50))。采用(0.04、0.08、0.16)μg/mL rflaA处理RAW264.7细胞24、36、48 h,采用ELISA检测IL-6、IL-1β分泌水平;(0.04、0.08、0.16)μg/mL rflaA处理RAW264.7细胞6、12、24、36、48 h,收集细胞,实时定量PCR检测IL-6、IL-1β、NOD样受体蛋白3(NLRP3)、胱天蛋白酶1(caspase-1)mRNA的含量;Western blot法检测NLRP3、caspase-1蛋白水平。结果 rflaA可促进RAW264.7细胞因子分泌,RAW264.7细胞被不同剂量的rflaA作用24、36、48 h,随rflaA剂量的递增,IL-6、IL-1β水平增加,以0.16μg/mL剂量时作用最显著,36 h达高峰;rflaA可增加RAW264.7细胞的IL-6、IL-1β、NLRP3、caspase-1 RNA水平,0.16μg/mL rflaA作用最显著,12 h达高峰;RAW264.7细胞被不同剂量的rflaA作用6、12、24、36 h,随rflaA剂量增加NLRP3、caspase-1表达水平增加,以0.16μg/mL剂量时作用最显著,24 h达高峰。结论 rflaA通过刺激NLRP3、caspase-1而增加RAW264.7细胞IL-6、IL-1β的分泌。
Objective To investigate the effect of recombinant Le pneumophila flagellin A (rflaA) on the secretion of interleukin 6 (IL-6) and IL-1β by RAW264.7 macrophages and its mechanism. Methods RAW264.7 cells were treated with rflaA (0.000,0.125,0.250,0.500,1.000,2.000,4.000,8.000) μg / mL, and the cell control group was established by CCK-8 to determine the half effect dose of rflaA (EC 50 )). RAW264.7 cells were treated with (0.04, 0.08, 0.16) μg / mL rflaA for 24, 36 and 48 h respectively, and the levels of IL-6 and IL-1β were detected by ELISA. RAW264.7 cells were treated with rflaA .7 cells were harvested at 6, 12, 24, 36, and 48 hours. The cells were harvested and the expression of IL-6, IL-1β, NORP3 and caspase-1 mRNA Content; Western blot detection of NLRP3, caspase-1 protein levels. Results rflaA could promote the secretion of RAW264.7 cytokine, and RAW264.7 cells were treated with different concentrations of rflaA for 24, 36 and 48 h. The levels of IL-6 and IL-1β increased with the increase of rflaA dose. RflaA increased the levels of IL-6, IL-1β, NLRP3 and caspase-1 RNA in RAW264.7 cells. The most significant effect of rflaA at 0.16μg / mL reached the peak at 12 h. 7 cells were treated with different dosage of rflaA for 6,12,24,36 h. With the increase of rflaA dosage, the expression level of NLRP3 and caspase-1 increased, reaching the peak at 0.16 μg / mL and reaching the peak at 24 h. Conclusion rflaA can increase the secretion of IL-6 and IL-1β in RAW264.7 cells by stimulating NLRP3 and caspase-1.