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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。
Objective To investigate the protein expression of immortalized human epidermal cells (HaCaT) induced by 15 nm silica in vitro using proteomic techniques. Methods HaCaT cells were treated with 2.5, 5.0 and 10.0 mg / L of 15 nm silica solution respectively for 24 h and dd H2O as solvent control. CCK-8 method was used to detect cell proliferation. The subcellular proteome extraction kit was used to extract membrane proteins from HaCaT cells and normal control cells treated with 10.0 mg / L 15 nm silica solution for 24 h. The differential 2D-gel electrophoresis (2D-DIGE) Matrix Assisted Laser Time of Flight (MALDI-TOF / TOF) mass spectrometry identification. The differential proteins were clustered by GO (gene oncology) function and predicted by transmembrane domain. Western blot was used to verify the differential protein expression. Results The results of cell proliferation assay showed that 15 nm silica could decrease the overall proliferation of HaCaT cells in a dose-dependent manner. Compared with normal cells, HaCaT cells were treated with 2.5, 5.0 and 10.0 mg / L of 15 nm silica 24 h, the levels of vitality were (91.3% ± 6.1%), (81.7% ± 7.0%) and (74.0% ± 2.6%), respectively, with statistical significance (P <0.05). Mass spectrometry identified ten differentially expressed proteins, of which seven differential proteins had more than one transmembrane domain. GO functional clustering indicated that these differential proteins were mainly involved in both binding activity and structural molecular activity. Western blot results showed that the trend of G protein-coupled receptor 179 and L-actin expression was consistent with that of proteomics analysis. Conclusion 15 nm silica can reduce the proliferation of HaCaT cells and lead to the decrease of membrane protein expression.