【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(P<0.05)。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。更多还原

【Abstract】 Objective To identify the differential expression of membrane proteins after HaCaT cell was treated with 15 nm silica nanomaterials( SiO2). Methods The HaCaT cells were cultured for 24 h under 15 nm SiO2 in various concentrations( 2. 5,5. 0,10. 0 mg / L),and dd H2O were used as control. The cell viability were measured with CCK-8 assay. The membrane proteins of SiO2-treated group( 10. 0 mg / L) and controls were extracted by Proteo Extract subcellular proteome extraction kit. The differentially expressed membrane proteins were analyzed by a two-dimensionalfluorescence difference gel electrophoresis( 2D-DIGE) and matrix-assisted laser desorption / ionization tandem time-of-flight mass spectrometry( MALDI-TOF-MS).Bioinformatics analysis was used to reveal the biological functions and predict transmembrane domains of differential expressed proteins. The expression of differential membrane proteins were measured by Western blot analysis. Results The cell viability was significantly decreased with the increases of 15 nm SiO2 exposure levels. After treatment with 2. 5,5. 0,10. 0 mg / L of 15 nm SiO2,the cell viability was assessed to( 91. 3% ± 6. 1%),( 81. 7% ± 7. 0%) and( 74. 0% ± 2. 6%) of control level( P <0. 05),respectively. In the proteomic analysis,a total of 10 proteins were identified as differential expression in the SiO2-treated simples compared with controls. Among these,7of these proteins were predicted as membrane proteins with at least one significant transmembrane domain. The most dominant function that the identified proteins involved in was binding and structural molecule activity. The differential expression of G proteincoupled receptor 179( GPR 179) and L-plastin( LCP1) were verified by Western-blot analysis under 15 nm SiO2 exposure in various concentrations. Conclusion The exposure of 15 nm SiO2 can significantly reduce the cell proliferation and induce a down-regulation of membrane protein expression in Ha Ca T cells.更多还原