5-Aza-CdR对人喉鳞癌Hep2细胞凋亡及蛋白质表达的影响

Study of 5-Aza-CdR on apoptosis and protein expression alteration in laryngeal squamous carcinoma Hep2

目的:探讨5-Aza-CdR对人喉鳞癌Hep2细胞凋亡及蛋白质表达的影响。方法:Hep2细胞分别经10-7mol/L 5-Aza-CdR和DMSO处理72h,应用Hoechst33258染色及流式细胞仪检测5-Aza-CdR对Hep2细胞凋亡的影响。抽提各组Hep2细胞总蛋白质,行双向电泳,采用PDQuest-7.0.1软件及MALDI-TOF质谱技术筛查5-Aza-CdR对Hep2细胞蛋白表达的影响,并通过Western blot对显著性差异蛋白质进行验证。结果:人喉鳞癌Hep2细胞在10-7mol/L 5-Aza-CdR处理72h后出现明显的细胞核致密浓染,Hep2细胞的凋亡率由对照组的(2.70±0.28)%增加到(13.96±0.41)%,具有统计学意义(P<0.05)。双向电泳筛查了包括S100A4在内的5种表达显著差异的蛋白质,Western blot进一步验证了5-Aza-CdR能明显上调喉鳞癌Hep2细胞中S100A4的表达,进而证明了双向电泳结果的可信性。结论:5-Aza-CdR介导的Hep2细胞部分蛋白质表达改变,直接或间接地参与了5-Aza-CdR诱导的Hep2细胞凋亡的发生,为5-Aza-CdR在喉鳞癌探讨性治疗中的机制研究奠定基础。

Objective： To explore the effect of 5-Aza-CdR on apoptosis and protein expression alteration in laryngeal squamous carcinoma Hep2.Methods： Hep2 cells were treated with 10^-7mol/L 5-Aza-CdR and DMSO respectively.Hoechst33258 staining and flow cytometry were used to detect the effect of 5-Aza-CdR on Hep2 cell apoptosis.Followed by total protein extraction,two dimensional gel electrophoresis and PDQuest-7.0.1 software together with MALDI-TOF mass spectrometry were used to detect the altered proteins in Hep2 cells.While,western bolts were used to confirm the significant difference.Results： Obvious nucleus dense staining were found in Hep2 cells treated with 10-7mol/L 5-Aza-CdR for 72 hours,and the apoptosis rate increased from（13.96±0.41）% to（2.70±0.28）%,compared with DMSO group,which was significantly different（P〈0.05）.Two dimensional gel electrophoresis detected 5 significantly difference expressed proteins including S100A4.Western bolts further confirmed that 5-Aza-CdR could significantly up-regulate S100A4 expression in Hep2 cells,which confirmed the two dimensional gel electrophoresis results in our study were credible.Conclusions： S everal protein expression alterations mediated by 5-Aza-CdR participate in Hep2 cell apoptosis induced by the same agent directly or indirectly,which providing theoretical fundament for the mechanism of 5-Aza-CdR exploring treatment in laryngeal squamous carcinoma.