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

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 510)检测THP-1细胞NF-κB p65亚基(NF-κB／p65)定位；Western-blot法测定THP-1细胞核蛋白中NF-κB／p65水平。结果 免疫荧光分析显示，异硫氰酸荧光素(FTTC)标记的NF-κB／p65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／p65主要分布于胞浆区，核区很少，而100μg/mlSiO2刺激30min后NF-κB／p65荧光标记集聚于核区，胞浆区少见。Westem-blot结果显示，对照组(0μg/mlSiO2)THP-1细胞核蛋白中有低水平NF-κB／p65表达,100μg/ml和200μg/ml SiO2作用15min和30min，THP-1细胞核蛋白中NF-κB／p65表达增加，并随着剂量的增加和时间的延长，NF-κB／p65水平相对增加。NF-κB／p65激活剂脂多糖(LPS)处理的THP-1细胞NF-κB／p65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。

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

OBJECTIVE: To study the effect of silicon dioxide (SiO(2)) on the activation of nuclear factor-kappaB (NF-kappaB) in THP-1 cell line. METHODS: THP-1 cells were incubated with a series of doses of SiO(2) (0, 100, 200 micro g/ml). The location of NF-kappaB p65 subunit (NF-kappaB/p65) in THP-1 cells was detected by immunofluorescence and laser scanning confocal microscope (LSCM). The expression of NF-kappaB/p65 in nuclei was measured by Western blot analysis. RESULTS: The majority of fluorescein isothiocyanate (FITC)-labelled NF-kappaB/p65 located in the nuclei 30 min after stimulation by 100 micro g/ml SiO(2), whereas the FITC-labelled NF-kappaB/p65 were mainly seen in the plasma of normal control cells. The expression of NF-kappaB/p65 in THP-1 nuclear protein was low in control group (0 micro g/ml SiO(2)) while it increased after stimulation by 100 micro g/ml SiO(2) and 200 micro g/ml SiO(2) for 15 min and 30 min. The level of NF-kappaB/p65 was comparatively increased with the increasing of doses and time. Lipopolysaccharides (LPS), an activator of NF-kappaB, had similar effect as SiO(2) on the activation of NF-kappaB/p65 in THP-1 cells. CONCLUSION: SiO(2) could activate and internalize NF-kappaB in the THP-1 cell line.