摘要:
目的 建立耐他莫昔芬(TAM)人乳腺癌细胞耐药细胞株T47D-TamR,比较其与亲本细胞株T47D在1sF-FDG细胞摄取率上的差异,并进行机制研究.方法 采用长时间逐步增加药物浓度冲击法,诱导建立耐TAM人乳腺癌细胞耐药细胞株T47D-TamR.利用MTT法测定耐药株的耐药指数(RI)及增殖能力.分别在不同细胞数、反应时间、18 F-FDG用量及葡萄糖浓度的实验条件下,测定T47D-TamR和T47D细胞的18F-FDG细胞摄取率.利用试剂盒检测2种细胞内LDH活性、ATP水平及上清液乳酸含量.通过Western blot法检测2组细胞中ERα和Glut-1的表达量及AMPK和mTOR的磷酸化水平.采用两样本t检验或双因素方差分析处理数据.结果 成功构建T47D-TamR细胞株,其RI为1.97±0.08,且增殖能力明显低于亲本细胞(F=230.10,P<0.05).分别改变细胞数、反应时间及18F-FDG用量时,T47D-TamR细胞的18F-FDG细胞摄取率均低于T47D细胞,2组间差异均有统计学意义(F值:419.00~1 116.00,均P<0.05).T47D和T47D-TamR细胞内LDH活性分别为(0.42±0.04)和(0.32±0.02) U/mg蛋白质,ATP水平分别为(19.99±0.32)和(14.01±0.70) nmol/mg蛋白质,上清液乳酸浓度分别为(2.95±0.05)和(2.02±0.07) mmol/L,2组间差异有统计学意义(t值:4.39~18.80,均P<0.05).T47D和T47D-TamR细胞中ERα、磷酸化AMPK(p-AMPK)、磷酸化mTOR (p-mTOR)、Glut-1的相对表达量分别为0.26±0.03、0.36±0.06、0.75±0.11、0.35±0.07和0.17±0.02、0.61±0.09、0.52±0.08、0.21±0.04,2组间差异有统计学意义(t值:12.20~16.45,均P<0.05).结论 T47D-TamR细胞的1s F-FDG摄取率、胞内LDH活性、ATP水平及上清液乳酸含量均低于T47D细胞,且其p-AMPK的表达量升高,ERα、p-mTOR、Glut-1的表达量降低,提示耐TAM乳腺癌细胞糖酵解水平降低.%Objective To establish the acquired tamoxifen (TAM)-resistant human breast cancer cell line T47D-TamR,compare the 18F-FDG uptake rate between T47D-TamR and its parental cell line T47D,and study the mechanism.Methods Long-term step-wise drug stimulation was used for cell line T47D-TamR establishment and then the cell proliferation and resistance index (RI) were determined by MTT assay.The 18F-FDG uptake rates of T47D-TamR and T47D cells were measured in the setting of different cell counts,reaction time,18F-FDG dosages and glucose concentrations.The LDH activity,cellular ATP level and lactic acid concentration in cell supernatant of T47D-TamR and T47D cells were detected.Western blot was used to examine the expression of ERα,Glut-1,phosphorylated AMPK (p-AMPK) and phosphorylated mTOR (p-mTOR).Two-sample t test and two-way analysis of variance were used to analyze the data.Results T47D-TamR cell line was successfully established and its drug RI was 1.97±0.08,with a significantly decreased cell proliferation efficacy (F =230.10,P< 0.05).Significant differences of 18 FFDG uptake rates were found between T47D-TamR cell and T47D cell when changing the cell count,reaction time,and 18F-FDG dosage (F values:419.00-1 116.00,all P<0.05).The LDH activities of T47D cell and T47D-TamR cell were (0.42±0.04) and (0.32±0.02) U/mg protein,cellular ATP levels were (19.99±0.32) and (14.01±0.70) nmol/mg protein,lactic acid concentrations in cell supematant were (2.95±0.05) and (2.02±0.07) mmol/L,respectively.The differences of above parameters between the two groups were significant (t values:4.39-18.80,all P<0.05).The relative expressions of ERα,p-AMPK,pmTOR,Glut-1 were 0.26±0.03,0.36±0.06,0.75±0.11,0.35±0.07 in T47D cell,and 0.17±0.02,0.61±0.09,0.52±0.08,0.21±0.04 in T47D-TamR cell,and there were significant differences (t values:12.20-16.45,all P<0.05) between the two groups.Conclusions Compared with parental cells,T47D-TamR cells have lower 18F-FDG uptake rate,LDH activity,cellular ATP level and lactic acid concentration,increased p-AMPK expression and decreased ERα,p-mTOR,Glut-1 expression,indicating the decreased glycolysis ability in TAM-resistant breast cancer cells.