rVvhA作用于THP-1细胞NF-κB信号通路的研究

Study on NF-κB Activation Pathway in rVvhA-effected THP-1 Cells

研究重组创伤弧菌溶细胞素(rVvhA)对人单核细胞系(THP-1)NF-κB信号通路的影响。应用CCK-8法检测rVvhA对THP-1细胞增殖的抑制作用;倒置显微镜和激光共聚焦显微镜观察rVvhA作用后细胞的形态学变化和细胞内NF-κB p65的核转移情况;应用流式细胞仪和Westernblot检测rVvhA作用后细胞浆内和细胞核内NF-κB p65的表达情况;ELISA检测rVvhA作用于细胞后TNF-α、IL-6表达含量的变化。试验结果显示:rVvhA能够呈时间–剂量依赖性地抑制THP-1细胞的生长,且镜下可见明显的细胞形态学变化。激光共聚焦显示0.4 HU/mL rVvhA作用6 h后,THP-1细胞内NF-κB p65的核转移现象最明显;流式细胞仪结果显示0.6 HU/mL rVvhA作用2 h后,细胞内总的NF-κB p65表达量达到高峰;Western blot检测显示0.6 HU/mL rVvhA作用4 h时细胞核内NF-κBp65蛋白含量最高;ELISA显示TNF-α的表达在rVvhA作用的适当范围内呈时间–剂量依赖性变化;IL-6在0.6 HU/mL rVvhA作用时表达量最高,随后随着浓度的增加反而下降;NF-κB抑制剂能使IL-6表达量下调。实验证明rVvhA作用于THP-1细胞后能激活NF-κB信号通路,上调TNF-α、IL-6的表达,而NF-κB抑制剂能够下调IL-6的表达。

This study designed to investigate how recombinant Vibrio vulnificus hemolysin(rVvhA) actives NF-κB signaling pathway in the PMA-stimulated THP-1 cell line.The cytotoxic effect of rVvhA on the growth of THP-1 cells was identified by CCK-8.Cell morphology was observed by inverted microscope.NF-κB p65 nuclear transfer was observed by laser scanning confocal microscope.NF-κB p65 expression in rVvhA effected THP-1 cells was detected by flow cytometry.NF-κB p65 expression in rVvhA effected THP-1 cell nucleus was determined by Western blot analysis.TNF-α and IL-6 expression in rVvhA effected THP-1 cells was quantified by ELISA.The viability of THP-1 cells exposed to rVvhA was inhibited in time-dose dependent mannar and the cell morphology changed obviously observed by inverted microscope.The levels of NF-κB p65 transferred into nuclear reached its tiptop after treated by 0.4 HU/mL rVvhA for 6 h.Total content of NF-κB p65 in cells peaked at 2 h treated by 0.6 HU/mL rVvhA.NF-κB p65 in cell nucleus was the most after 0.6 HU/mL rVvhA treated for 4 h.rVvhA effected THP-1 cells can raise the expressions of TNF-α and IL-6.NF-κB inhibitors can restrain the expression of IL-6.rVvhA had cytotoxic effect on THP-1 cells.It can activate NF-κB signaling pathway in THP-1 cells and increase the expressions of TNF-α and IL-6,meanwhile,NF-κB inhibitors can decrease the expression level of IL-6.