【摘要】 目的:探讨细胞外信号调节激酶(ERK1/2)通路、多药耐药基因(mdr-1)、核糖核苷还原酶M1(RRM1),在胰腺癌细胞株SW1990吉西他滨(GEM)化疗耐药中的相互关系及作用。方法:通过浓度梯度递增法,诱导建立耐各种浓度GEM的胰腺癌细胞株。应用免疫组化图像分析方法,半定量检测各耐药细胞株中ERK1/2蛋白的表达;RT-PCR的方法检测mdr-1、RRM1mRNA表达,条带图使用Band凝胶分析软件进行半定量分析;MTT法测定OD492光密度值,计算IC50值。结果:建立的耐药细胞株可以在GEM终浓度分别为0、30、60、100、150、200nmol/L的培养液中稳定生长并传代。ERK1/2、mdr-1、RRM1的表达均随着耐GEM浓度的增加而升高,细胞耐GEM的浓度分别与mdr-1、RRM1基因表达呈现出高度正相关(r=0.960,P=0.002和r=0.966,P=0.002);ERK1/2灰度值也与mdr-1、RRM1基因的表达存在相关性(r=-0.943,P=0.005和r=-0.883,P=0.02)。耐GEM200nmol/L的细胞株,ERK1/2灰度值、mdr-1/β-actin、RRM1/β-actin分别为164.22±13.17、1.41±0.04、1.45±0.18;经阻断剂U0126作用后三者的表达均呈现同步降低,分别为186.85±13.14、0.23±0.02、0.21±0.03;而激活剂EGF作用后,其表达呈现升高趋势,分别为106.55±16.45、1.50±0.07、1.52±0.12。耐GEM0nmol/L和200nmol/L浓度的细胞株,其IC50值分别为4.104和10.20,经阻断剂U0126处理后,IC50值下降到3.26和4.50;而经EGF处理后,IC50值则分别上升到8.89和17.17。结论:ERK通路可能参与调控mdr-1、RRM1基因表达,从而介导胰腺癌细胞株SW1990吉西他滨化疗抵抗。更多还原

【Abstract】 To investigate the role of extracellular signal-regulated kinase pathway (ERK), multidrug resistance 1 (MDR-1) and ribonucleoside reductase M1 (RRM1) in Gemcitabine (GEM) chemoresistance in the pancreatic cancer cell line SW1990. Methods: The GEM-resistant pancreatic cancer cell line model was constructed using a stepwise increase in concentration gradient of Gemcitabine. Immunocytochemistry was performed to detect the expression of ERK1/2 in the established cell lines in a semiquantitative way, and the mRNA expression of MDR-1 and RRM1 in the GEM-resistant pancreatic cancer cell lines was detected by RT-PCR. MTT assay was used to determine the IC50. Results: The established pancreatic cancer cell lines cultured in medium with GEM at different concentrations (0, 30, 60, 100, 150 and 200 nmol/L) were able to grow and maintain their resistance through several passages. The expression of ERK1/2, MDR-1 and RRM1 were elevated according to the increasing GEM concentration. There was a highly positive correlation between MDR-1 or RRM1 expression and GEM concentration (r=0.960,P=0.002 and r=0.966, P=0.002). The gray scale of ERK1/2 was also correlated with the expression of MDR-1 and RRM1 (r=-0.943, P=0.005 and r=-0.883,P=0.02). At a concentration of 200 nmol/L, the gray scale of ERK1/2, MDR-1/β-actin, and RRM1/β-actin of the GEM-resistant pancreatic cancer cell line was 164.22±13.17, 1.41±0.04 and 1.45±0.18, respectively. The expression of these genes decreased synchronously after the cellswere treated with ERK1/2 signaling pathway specific inhibitor U0126 to the gray scale values of 186.85±13.14, 0.23±0.02 and 0.21±0.03, respectively. Conversely, there was a tendency to decreased expression of these genes after the cells were treated with epidermal growth factor (EGF). The gray scale values ascended to 106.55±16.45, 1.5±0.07 and 1.52±0.12, respectively. The IC50 of the GEM-resistant pancreatic cancer cell lines at the concentration of 0 nmol/L and 200 nmol/L were 4.104 and 10.20, respectively. There was a decrease in the IC50 to 3.26 and 4.5 after the cells were treated with U0126, and an elevation to 8.89 and 17.17 after the cells were treated with EGF. Conclusion: The ERK signaling pathway participates in the regulation of the expression of MDR-1 and RRM1, most likely contributing to the GEM chemoresistance in the SW1990 pancreatic cancer cell line.更多还原