摘要目的 利用蛋白质组学方法,研究经或不经姜黄素处理的人结肠癌耐药细胞之间差异表达的蛋白,以发现与结肠癌耐药相关的蛋白.方法 以固相pH梯度等电聚焦为第一向,垂直平板十二烷基磺酸钠聚丙烯酰胺凝胶电泳为第二向分离经姜黄素处理和对照组人结肠癌耐长春新碱细胞系(HCT-8/VCR)总蛋白,通过凝胶银染显色,扫描仪GS-800采集凝胶图像,图像分析软件PDQuest8.0分析电泳图谱寻找有意义的差异蛋白点,运用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)系统对选取得蛋白点进行质谱鉴定,RT-PCR和Western印迹对谷胱苷肽S-转移酶P1(GSTP1)蛋白质进行验证.结果 获得了背景清晰、分辨率高、重复性好的HCT-8/VCR细胞系2-DE图谱,姜黄素组和对照组图像的平均蛋白质点数分别为1 030±69和1 070±96;两者的差异点共29个,选取其中一个蛋白并用质谱分析证实其为GSTP1.RT-PCR、Western印迹结果显示姜黄素能够下调GSTP1 mRNA和GSTP1蛋白的表达,姜黄素组GSTP1 rnRNA (0.49±0.09)和GSTP1蛋白(0.29±0.07)表达明显低于对照组GSTP1 mRNA(1.19±0.21)和GSTP1蛋白(0.70±0.13)表达(均P＜0.05).结论 姜黄素可能通过抑制GSTP1蛋白的表达来增强HCT-8/VCR对长春新碱的化疗敏感性,GSTP1蛋白质可能参与姜黄素逆转结肠癌细胞长春新碱耐药过程.

abstractsObjective To explore the proteomic differences among with and without curcumin treatment of vincristine-resistance HCT-8/VCR cells of human colon carcinoma by using mass spectrometry and two-dimensional gel electrophore-sis (2-DE).Methods The total proteins of the both groups were extracted from serum were run in immobilized pH gradient isoelectic focusing (IPG-IEF) at the first dimension.The proteins pots in gels were visualized by silver staining protocol,scanned by using a molecular imager GS-800 calibrated densitometer.The differentially expressed proteins were identified and analyzed by PDQuest 8.0 software.The diferentially displayed protein spots were searched and identifiyed by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS),the interested proteins were further validated by RT-PCR and Western blot.Results The 2-DE HCT-8/VCR cells patterns were acquired with clear background,well-resolution and reproduction.And 1 070 ± 96 protein spots were detected in control HCT-8/VCR cells and 1 030 ± 69 in curcumin-treated HCT-8/VCR cells.Twenty-nine differential protein spots were found to be differentially expressed.Glutathione S-transferase pi1 gene (GSTP1),a diferentiaI expression protein was identified which one of these proteins.RT-PCR and Western blotting results showed that the expressions of GSTP1 mRNA (0.49 ± 0.09) and protein (0.29 ± 0.07) in curcumin-treated group were significantly lower than in control group(GSTP1 mRNA 1.19 ± 0.21 and protein 0.70 ± 0.13,both P ＜ 0.05),indicating that curcumin down regulated these expressions.Conclusions The suppression of GSTP1 by curcumin could enhance the vincristine chemosensitivity in HCT-8/VCR.GSTP1 overexpression may be involved in the vincristine-resistance of human colon carcinoma cells.