应用蛋白质组学技术筛选胃癌耐药相关蛋白质

胃癌多药耐药性是临床胃癌化疗失败最主要的原因之一,但其分子机制仍然不太清楚.为了寻找新的胃癌耐药相关的蛋白质,揭示胃癌多药耐药的分子机制,以胃癌细胞SGC7901和长春新碱诱导的耐药胃癌细胞SGC7901/VCR为研究对象,应用二维凝胶电泳(two-dimensionalelectrophoresis,2-DE)技术分离两种细胞的总蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)及电喷雾电离串联质谱(electrosprayionizationtandemmassspectrometry,ESI-Q-TOF)对差异表达的蛋白质点进行鉴定,蛋白质印迹和实时RT-PCR验证部分差异蛋白质在两株细胞中的表达水平,反义核酸转染技术分析HSP27(heatshockprotein27,HSP27)高表达与SGC7901/VCR耐药的相关性.得到了分辨率较高、重复性较好的两株细胞系的二维凝胶电泳图谱,质谱分析共鉴定了24个差异蛋白质点,蛋白质印迹和实时RT-PCR验证了部分差异蛋白的表达水平,反义寡核苷酸抑制HSP27表达能增加SGC7901/VCR对长春新碱的敏感性.研究结果不仅提示这些差异蛋白质如HSP27,Sorcin等可能与胃癌的多药耐药相关,而且为揭示胃癌细胞的多药耐药性产生机制提供了线索.

Identification of Multidrug Resistance Related Proteins in Human Gastric Cancer Cells Using Proteomics Technique

Resistance to anticancer drugs is one major problem preventing effective chemotherapy of gastric cancer, but the molecular mechanisms of multidrug resistance (MDR) of gastric cancer is not completely clear. In order to find out new MDR related proteins of gastric cancer, two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of vincristine-resistant human gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901, PDQuest software was applied to analyze 2-DE images, and the differential protein spots between the two cell lines were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Then the differential expressional levels of partial identified proteins were detected by Western blot analysis and real-time RT-PCR. And the effect of HSP27 on the development of MDR of SGC7901/VCR was determined by antisense oligonucleotides (ASO) technique. The well-resolved, reproducible 2-DE patterns of SGC7901/VCR and SGC7901 were established. All the 24 differential proteins were identified, and the differential expression levels of the partial proteins between the two cell lines were confirmed by Western blot analysis and real-time RT-PCR. The suppression of HSP27 expression by HSP27 ASO could enhance vincristine chemosensitivity in SGC7901/VCR. These differential proteins such as HSP27 and sorcin may be related to MDR in SGC7901/VCR cells. The data will be valuable for further to study the mechanism of MDR in human gastric cancer.