【摘要】 目的探讨全氟化碳对内毒素诱导的人肺泡Ⅱ型上皮细胞(A549细胞)炎症因子TNF-α表达的影响及机制。方法人肺泡Ⅱ型上皮细胞,传代培养后,均分为3组:空白对照组(C组)、脂多糖(LPS)组、全氟化碳+LPS(P+LPS组),P+LPS组在给予LPS同时加入全氟化碳,LPS的终浓度为10μg/ml,全氟化碳的终浓度为12.5%,各组分别孵育30 min、2 h、4 h、12 h、24 h,结束后用蛋白免疫印迹法(Western blot)检测核转录因子-κB(NF-κB)、Toll样受体4(TLR4)蛋白表达水平,酶联免疫吸附法(ELISA)检测细胞培养上清中TNF-α的水平。结果 (1)Western blot结果显示,C组人肺泡Ⅱ型上皮细胞上有TLR4蛋白的表达,但表达量较低;LPS组人肺泡Ⅱ型上皮细胞在30 min、2 h、4 h、12 h、24 h点TLR4及NF-κB蛋白的表达量明显高于C组(P<0.05);与LPS组比较,P+LPS组人肺泡Ⅱ型上皮细胞30 min、2 h、4 h、12 h、24 h点TLR4及NF-κB蛋白表达明显下降(P<0.05),C组人肺泡Ⅱ型上皮细胞的TLR4及NF-κB蛋白表达量与P+LPS组相比,无明显差异(P>0.05)。(2)ELISA结果显示,与C组比较,LPS组人肺泡Ⅱ型上皮细胞30 min、2 h、4 h、12 h、24 h点TNF-α均明显升高(P<0.05)。P+LPS组人肺泡Ⅱ型上皮细胞30 min、2 h、4 h、12 h、24 h点TNF-α较LPS组均明显降低(P<0.05)。结论 (1)TLR4可能介导了LPS诱导的人肺泡Ⅱ型上皮细胞的炎症反应。(2)全氟化碳减轻LPS诱导的人肺泡Ⅱ型上皮细胞的炎症反应,可能与抑制细胞表面TLR4的信号活化,进一步抑制下游NF-κB的转导路径,从而降低TNF-α的产生有关。更多还原

【Abstract】 Objective To investigate the molecular mechanism of perfluorocarbon in lipopolysaccharide(LPS)-induced signal pathways in A549 cells.Methods A549 cells(AT-Ⅱ cells),cultured,were divided into 3 groups:control group(C),group LPS(L),perfluorocarbon+LPS(P+LPS),AT-Ⅱ cells of P+LPS group were cultured with LPS and perfluorocarbon simultaneously,final concentration of LPS 10 μg/ml,final concentration perfluorocarbon 12.5%.AT-Ⅱ cells of each group were incubated and examined at 30 min,2 h,4 h,12 h,and 24 h.TNF-α in supernatant of four groups were determined by enzyme linked immunosorbent assay(ELISA),NF-κB and TLR4 determined by Western blot.Results(1)TLR4 and NF-κB protein in group LPS at 30 min,2 h,4 h,12 h,and 24 h were significantly higher than those in group C(P<0.05);Then decreased in group P+LPS significantly(P<0.05).(2)TNF-α in LPS group at 30 min,2 h,4 h,12 h,and 24 h were significantly higher than those in group C(P<0.05) and decreased significantly in P+LPS group(P<0.05).Conclusion(1)TLR4 may mediate inflammatory reaction induced by LPS in AT-Ⅱcells.(2)Perfluorocarbon can alleviate inflammatory reaction induced by LPS in AT-Ⅱ cells by suppressing signal activation of TLR4 on the cell surface and signal transduction of downstream NF-κB,thereby reducing the production of TNF-α.更多还原