人胰腺癌细胞中对5-氟尿嘧啶耐药相关基因的检测分析

Identification of genes associated with 5-fluorouracil resistance in human pancreatic cancer cells

目的:利用基因芯片技术,对体外筛选获得的耐不同浓度5-氟尿嘧啶(5-FU)的胰腺癌细胞株基因表型进行对比,找出不同耐药表型细胞株的基因表达改变情况,以此发现一些与胰腺癌细胞5-FU耐药相关的重要基因或通路。方法:用不断增加浓度的5-FU反复刺激细胞,直到细胞能完全耐受不同浓度5-FU,获得耐不同浓度5-FU的MIAPaCa-2细胞株。由基因芯片筛选出明显改变的基因后再用实时定量PCR方法验证。结果:本研究成功地获得两种对5-FU耐药的胰腺癌细胞株,低耐药表型MIA-FU-2.4和高耐药表型MIA-FU-10.0。基因分析结果提示耐药可能与广泛的基因表达改变相关。在MIA-FU-2.4和MIA-FU-10.0两种表型中分别有1075及1628个基因出现了表达改变,涉及细胞周期、细胞黏附、信号转导、DNA修复和凋亡等多种生物学功能。结论:胰腺癌MIAPaCa-2细胞株5-FU耐药的发生涉及多种细胞生物学功能改变,特别与凋亡、DNA修复、细胞周期等基因和一些信号转导途径相关。

Objective：Using gene expression data,we compared the genotype expression profile of different 5-fluorouracil （5-FU） resistance phenotypes of pancreatic cancer ceils after in vitro selection. The aims of our study were to identify genes showing altered expression in resistant derivatives,as well as several central genes or important pathways that were associated with 5-FU resistance in pancreatic cancer cells. Methods：The 5-FU-resistant MIAPaCa-2 derivatives were developed through exposure to increasing concentrations of 5-FU with repeated subcultures until the cells became fully resistant to 5-FU. Gene array analysis was performed using a Uniset Human 20 K I Codelink Bioarray. Data were analyzed with CodeLink System Software. The expression profiling of selected genes was confirmed by real-time PCR assays. Results：We got successively two resistant derivatives：the low-resistance phenotype MIA-FU-2.4 and the high-resistance phenotype MIA-FU-IO.O. Analysis of array data showed that the resistance of 5-FU in pancreatic cancer cell was related to widespread transcriptional activation. In MIA-FU-2.4 and MIA-FU-10.0,we identified respectively 1075 and 1628 differentially expressed genes. The biological functions of these genes included cell cycle, cell adhesion, signal transduction, DNA repair and apoptosis et al. Conclusions： Our data suggest that 5-FU resistance development in pancreatic cancer cell MIAPaCa-2 involves in many aspects of the biological functions and might be mainly attributed to apoptosis, DNA repair and cell cycle mechanisms and some signal transductions or some pivotal genes.