纳米银对皮肤细胞半通道活性的影响及其在细胞增殖抑制中的作用

Effects of silver nanoparticle on hemichannel activation and anti-proliferation in HaCaT cells

目的:探讨纳米银(silver nanoparticles,AgNPs)对皮肤细胞半通道活性的影响,并进一步研究半通道活性的改变在AgNPs诱导的细胞增殖抑制中的作用。方法:不同浓度(0、0.1、0.2、1.0、5.0、10.0、15.0和20.0μg/cm^2)的AgNPs处理人永生化表皮细胞系(HaCaT)24 h,采用碘化丙啶(propidium iodide,PI)染料排除法测定细胞活性,采用溴化乙锭(ethidium bromide,EB)摄取法测定细胞半通道活性。通过以常规密度(4×10~4个/cm^2)或低密度(1×10~4个/cm^2)接种细胞及采用半通道抑制剂甘珀酸(carbenoxolone,CBX)预处理的方法调节细胞半通道活性,采用细胞增殖-毒性检测试剂盒(cell counting kit-8,CCK-8)检测半通道活性受抑制、不同浓度AgNPs作用后的HaCaT细胞的增殖水平。结果:当AgNPs浓度低于10μg/cm^2时,HaCaT细胞的细胞活性未受明显影响。10μg/cm^2的AgNPs作用2、12和24 h可导致细胞半通道活性升高至对照组的116.67%、124.85%和139.53%,与对照组相比差异均具有统计学意义(P<0.01),且25、50和100μmol/L CBX预处理可明显抑制10μg/cm^2AgNPs作用24 h后的细胞的半通道活性(P<0.01)。此外,100μmol/L CBX预处理可升高低密度和常规密度下10μg/cm^2AgNPs作用24 h后细胞的增殖水平,其中,低密度下,CBX预处理组与AgNPs单独处理组相比,细胞增殖水平显著升高(P<0.01)。结论:一定浓度的AgNPs可激活HaCaT细胞的半通道,半通道活性升高在AgNPs引发的细胞增殖抑制效应中具有重要作用。

Objective: To investigate the effect of silver nanoparticles( AgNPs) on hemichannel activity in human skin keratinocytes( HaCaT) and to explore the role of hemichannel in AgNP-induced antiproliferative effect on HaCaT cells. Methods: HaCaT cells were exposed to 0,0. 1,0. 2,1. 0,5. 0,10. 0,15. 0 and 20. 0 μg/cm^2 of AgNPs for 24 h and cell viability was assessed by propidium iodide( PI) staining with flow cytometry. Hemichannel activity was examined by ethidium bromide( EB) uptake experiments in cells exposed to AgNPs with and without hemichannel inhibitor carbenoxolone( CBX). Afterward,HaCaT cells were seeded at a low density of 1 × 104cell/cm^2 or a normal density of4 × 104cell/cm^2 and cultured for 24 h. Cell proliferation was measured by cell counting kit-8( cck-8) in low-or normal-density cultured cells exposed to AgNPs for 24 h with and without 100 μmol/L CBX. Results: Cell viability showed no significant differences between the control and AgNP-exposed groups with the concentration less than 10 μg/cm^2. Exposure to AgNPs increased EB uptake in a time-and dosedependent manner in HaCaT cells and EB fluoresce density was increased to 116. 67%,124. 85% and139. 53% of the control after exposure to 10 μg/cm^2 AgNPs for 2 h,12 h and 24 h. After being treated with 25,50 and 100 μmol/L CBX,10 μg/cm^2 AgNP-induced increase in EB uptake was significantly reduced in HaCaT cells( P < 0. 01). The cellular proliferation rate was increased inlow-and normaldensity cultured cells after AgNPs exposure with 100 μmol/L CBX. After being treated with 100 μmol/L CBX,10 μg/cm^2 AgNP-induced anti-proliferation in low-density cultured cells was significantly restrained( P < 0. 01). Conclusion: AgNPs could enhance hemichannel activity of HaCaT cells.Hemichannel activation was involved in AgNP-induced anti-proliferative effect.