二氧化硅致人单核细胞THP-1核因子-κB活化定位改变的研究

The effect of silicon dioxide on the activation of nuclear factor-κB in THP-1 cells

目的 探讨矽肺发病过程中SiO2 诱导对人单核细胞株THP 1核因子 κB(NF κB)活化的影响。方法 采用间接免疫荧光细胞化学法结合激光共聚焦显微镜 (LSCM ,Zeiss 5 10 )检测THP 1细胞NF κBp6 5亚基 (NF κB/p6 5 )定位 ;Western blot法测定THP 1细胞核蛋白中NF κB/p6 5水平。结果 免疫荧光分析显示 ,异硫氰酸荧光素 (FITC)标记的NF κB/p6 5在胞浆区为绿色荧光 ,而进入了丙亚基碘(PI)红色荧光标记的胞核区时则红绿两色叠加成黄色荧光。正常细胞NF κB/p6 5主要分布于胞浆区 ,核区很少 ,而 10 0 μg/mlSiO2 刺激 30min后NF κB/p6 5荧光标记集聚于核区 ,胞浆区少见。Western blot结果显示 ,对照组 (0 μg/mlSiO2 )THP 1细胞核蛋白中有低水平NF κB/p6 5表达 ;10 0 μg/ml和 2 0 0μg/mlSiO2 作用 15min和 30min ,THP 1细胞核蛋白中NF κB/p6 5表达增加 ,并随着剂量的增加和时间的延长 ,NF κB/p6 5水平相对增加。NF κB/p6 5激活剂脂多糖 (LPS)处理的THP 1细胞NF κB/p6 5定位情况和核蛋白水平与上述结果相似。结论 SiO2 刺激可引起THP 1单核细胞NF κB核转位活化。

Objective To study the effect of silicon dioxide(SiO_2) on the activation of nuclear factor-κB(NF-κB) in THP-1 cell line. Methods THP-1 cells were incubated with a series of doses of SiO_2(0,100,200 μg/ml).The location of NF-κB p65 subunit(NF-κB/p65) in THP-1 cells was detected by immunofluorescence and laser scanning confocal microscope(LSCM).The expression of NF-κB/p65 in nuclei was measured by Western blot analysis. Results The majority of fluorescein isothiocyanate(FITC)-labelled NF-κB/p65 located in the nuclei 30 min after stimulation by 100 μg/ml SiO_2,whereas the FITC-labelled NF-κB/p65 were mainly seen in the plasma of normal control cells.The expression of NF-κB/p65 in THP-1 nuclear protein was low in control group(0 μg/ml SiO_2) while it increased after stimulation by 100 μg/ml SiO_2 and 200 μg/ml SiO_2 for 15 min and 30 min.The level of NF-κB/p65 was comparatively increased with the increasing of doses and time.Lipopolysaccharides (LPS),an activator of NF-κB,had similar effect as SiO_2 on the activation of NF-κB/p65 in THP-1 cells. Conclusion SiO_2 could activate and internalize NF-κB in the THP-1 cell line.