胞质-5＇-核苷酸酶-Ⅱ基因与乳腺癌细胞对吉西他滨耐药的关系

Relationship on chemoresistance between the cytosolic 5＇-nucleotidase Ⅱ in breast cancer with induction of gemcitabine

目的 检测人乳腺癌MCF-7细胞株经吉西他滨处理时胞质-5＇-核苷酸酶-Ⅱ（CN-Ⅱ）mRNA和蛋白表达的变化,探讨其在乳腺癌化疗耐药中所起的作用.方法 以50 nmol/L吉西他滨干预MCF-7细胞,流式细胞仪测定0、3、7、10、13、16d细胞凋亡比例,并以实时定量聚合酶链反应（Real-time PCR）及Western blot方法测定存活细胞CN-Ⅱ的mRNA及蛋白表达.结果 干预后第0、3、7、10、13、16 d细胞中CN-Ⅱ的mRNA表达水平分别为1.00±0.21、1.06±0.31、1.11 ±0.29、2.15±0.41、2.46±0.72、3.58±1.34,与干预天数呈正相关（r=0.979）,蛋白表达水平分别为1.00±0.31、0.99±0.46、1.03 ±0.42、3.56 ±0.71、4.10±1.25、7.02±1.67,与干预天数呈正相关（r=0.971）,两者与凋亡细胞比例的变化趋势一致,相对耐药细胞中的CN-ⅡmRNA和蛋白表达水平与非耐药细胞比较,差异有统计学意义（P＜0.05）.结论 CN-Ⅱ在乳腺癌化疗时表达明显增强,可能与化疗耐药性的产生有关.

Objective To investigate the changes of 5＇-nucleotidase Ⅱ （CN-]Ⅱ） expression in human breast cancer cell line MCF-7 after induction of gemcitabine and study the effects of gemcitabine on chemoresistance in breast cancer.Methods After MCF-7 cells were incubated with 50 nmol/L gemcitabine for 0, 3, 7, 10, 13 and 16 days, the apoptosis ratio was determined by flow cytometry, and the mRNA and protein levels of CN-Ⅱ in the surival cells were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting, respectively.Results After MCF-7 cells were incubated with 50 nmol/L gemcitabine for 0, 3, 7, 10, 13 and 16 days, the expression of CN-lⅡ was 1.00 ± 0.21, 1.06 ± 0.31, 1.11 ± 0.29, 2.15 ± 0.41, 2.46 ± 0.72, and 3.58 ± 1.34 at mRNA level, which was positively correlated with incubation time （r =0.979） and consistent with the propprtion of apototic cells.The expression of CN-Ⅱ was 1.00 ±0.31, 0.99 ±0.46, 1.03 ±0.42, 3.56 ±0.71, 4.10 ± 1.25, and 7.02 ± 1.67 at protein level, which was positively correlated with incubation time （r =0.971） and consistent with the propprtion of apototic cells.When the drug resistant cells were compared with the non-drug resistant cells, the expression of mRNA and protein of CN-Ⅱ and APE/Ref-1 showed statistically significant difference.（P 〈 0.05）.Conclusion The obviously up-regulated expression of CN-Ⅱ and APE/Ref-1 may be an adaptive response contributing to the overall chemoresistance in breast cancer.