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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。
To investigate the effect of recombinant Vibrio parahaemolyticus (rVvhA) on NF-κB signaling pathway in human monocytic cell line (THP-1). The inhibitory effect of rVvhA on the proliferation of THP-1 cells was detected by CCK-8 assay. The morphological changes of rVvhA-treated cells and the nuclear translocation of NF-κB p65 were observed by inverted microscope and laser scanning confocal microscopy. Flow cytometry Western blot was used to detect the expression of NF-κB p65 in the cytoplasm and nucleus after rVvhA treatment. The changes of TNF-α and IL-6 expression after rVvhA treatment were detected by ELISA. The results showed that: rVvhA can inhibit the growth of THP-1 cells in a time-dose-dependent manner, and obvious morphological changes of cells were observed. Laser confocal microscope showed that nuclear translocation of NF-κB p65 in THP-1 cells was the most obvious after treated with 0.4 HU / mL rVvhA for 6 h. The results of flow cytometry showed that after 2 h of 0.6 HU / mL rVvhA treatment, The expression of NF-κB p65 peaked. Western blot showed that NF-κB p65 protein content was the highest in the cells treated with 0.6 HU / mL rVvhA for 4 h. ELISA showed that the expression of TNF-α was in a dose-dependent manner in the proper range of rVvhA The expression of IL-6 was the highest when treated with 0.6 HU / mL rVvhA, and then decreased with the increase of concentration. IL-6 expression was down-regulated by NF-κB inhibitor. Experiments show that rVvhA can activate NF-κB signaling pathway and up-regulate the expression of TNF-α and IL-6 after THV-1 cells, while NF-κB inhibitor can down-regulate the expression of IL-6.