纳米二氧化硅致人永生化表皮细胞膜蛋白质组的影响

发布时间：2018-03-13 16:43

本文选题：纳米二氧化硅　切入点：人永生化表皮细胞　出处：《卫生研究》2015年05期 　论文类型：期刊论文

【摘要】：目的采用蛋白质组学技术探讨15 nm二氧化硅致体外培养的人永生化表皮细胞(HaCaT)膜蛋白质表达的变化。方法分别以2.5、5.0和10.0 mg/L的15nm二氧化硅溶液处理HaCaT细胞24 h,以dd H2O为溶剂对照。应用CCK-8方法检测细胞增殖情况。运用亚细胞蛋白质组提取试剂盒对10.0 mg/L的15 nm二氧化硅溶液处理24 h的HaCaT细胞和正常对照组细胞进行膜蛋白提取,采用差异荧光双向凝胶电泳(2D-DIGE)分析和基质辅助激光解析飞行时间(MALDI-TOF/TOF)质谱鉴定。对差异蛋白质进行GO(gene oncology)功能聚类及跨膜结构域预测分析,采用Western blot免疫印迹验证差异蛋白的表达。结果细胞增殖实验显示,15 nm二氧化硅可以引起HaCaT细胞整体增殖水平下降,呈剂量依赖关系;与正常细胞相比,HaCaT细胞经15 nm二氧化硅以2.5、5.0和10.0 mg/L处理24 h后,活力水平分别为(91.3%±6.1%)、(81.7%±7.0%)和(74.0%±2.6%),差异有统计学意义(P0.05)。质谱鉴定了10个差异表达蛋白,其中7个差异蛋白具有1个以上的跨膜结构域,GO功能聚类表明这些差异蛋白主要涉及结合活性和结构分子活性。Western blot免疫印迹结果表明G蛋白偶联受体179和L-肌动蛋白表达变化趋势与蛋白质组学分析结果一致。结论 15 nm二氧化硅能够引起HaCaT细胞增殖水平降低,并且导致膜蛋白表达水平的下降。
[Abstract]:Objective to investigate the changes of protein expression in cultured human immortalized epidermal cells induced by 15nm silica by proteomics. Methods HaCaT cells were treated with 2.5 mg/L and 10.0 mg/L of 15nm silica solution for 24 h and then treated with dd for 24 h. H _ 2O was used as solvent control. CCK-8 method was used to detect cell proliferation. Membrane protein was extracted from HaCaT cells treated with 10.0 mg/L 15 nm silica solution for 24 h and normal control cells. Differential fluorescence two-dimensional gel electrophoresis (2D-DIGE) analysis and MALDI-TOF / TOF mass spectrometry were used to identify the differential proteins. The differential proteins were clustered by GO(gene oncology and predicted by transmembrane domain. Western blot Western blot was used to detect the differential protein expression. Results Cell proliferation assay showed that 15 nm silica could induce the overall proliferation of HaCaT cells to decrease in a dose-dependent manner. Compared with normal cells, the activity levels of HaCaT cells were 91.3% 卤6.1% 卤7.0% and 74.0% 卤2.6% respectively after treated with 15nm silica for 24 h. Ten differentially expressed proteins were identified by mass spectrometry. Seven of the differential proteins have more than one transmembrane domain go functional cluster. These differential proteins are mainly involved in binding activity and structural molecular activity. Western blot Western blot results show that G protein coupled receptors 179 and L- actin. Conclusion 15 nm silica can reduce the proliferation of HaCaT cells. It also leads to the decrease of membrane protein expression level.
【作者单位】：暨南大学药学院;深圳市疾病预防控制中心
【基金】：国家自然科学基金(No.21102094) 广东省卫生计生委科研项目(No.20132016;No.A2014659)
【分类号】：R114

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