论文部分内容阅读
目的:通过体外诱导耐药建立肺腺癌A549细胞系的培美曲塞耐药细胞株,并进一步研究其耐药性发生与HER2/neu之间的关系。方法:通过高浓度反复间歇诱导法建立A549的培美曲塞耐药细胞系。CCK8法检测耐药性、绘制生长曲线,计算倍增时间;流式细胞术法检测细胞周期分布及细胞凋亡水平;RT-PCR法检测耐药株及亲本株HER2/neu的mRNA表达。结果:历经6个月建立了耐药指数为6.7倍的A549/PEM细胞系,其较亲本株的倍增时间延长(33.5±1.71h vs 27.1±1.15h);流式细胞术检测结果显示:与亲本株比较,A549/PEM处于G1期的细胞比例增加(66.03±1.61%vs 57.92±1.73%),S期细胞比例降低(30.05±1.25%vs 37.51±1.31%),差别均有统计学意义(P<0.05);在PEM作用24h后,A549与A549/PEM细胞凋亡率分别为23.52%和10.99%,差别有统计学意义(P<0.05)。RT-PCR检测结果示:A549/PEM较亲本株HER2/neu基因的mRNA表达水平增高(P<0.05)。结论:A549/PEM与亲本株的生物学特性有差别;肺腺癌培美曲塞继发性耐药的产生可能与HER2/neu表达上调有关。
OBJECTIVE: To establish a pemetrexed-resistant cell line of human lung adenocarcinoma A549 cell line by inducing drug resistance in vitro and to further investigate the relationship between drug resistance and HER2 / neu. Methods: The A549 pemetrexed-resistant cell line was established by repeated intermittent induction with high concentration. CCK8 assay was used to detect the drug resistance, the growth curves were plotted and the doubling time was calculated. The cell cycle distribution and apoptosis were detected by flow cytometry. The mRNA expression of HER2 / neu was detected by RT-PCR. Results: The A549 / PEM cell line with a resistance index of 6.7 times was established after 6 months and the doubling time of the A549 / PEM cell line was prolonged (33.5 ± 1.71h vs 27.1 ± 1.15h) compared with the parent strain. The results of flow cytometry Compared with the parental strain, the proportion of A549 / PEM cells in G1 phase increased (66.03 ± 1.61% vs 57.92 ± 1.73%) and the proportion of S phase cells decreased (30.05 ± 1.25% vs 37.51 ± 1.31%), the difference was statistically significant P <0.05). After treated with PEM for 24 h, the apoptosis rates of A549 and A549 / PEM cells were 23.52% and 10.99%, respectively, with statistical significance (P <0.05). The results of RT-PCR showed that the mRNA expression of HER2 / neu gene in A549 / PEM was higher than that in parental strain (P <0.05). CONCLUSION: The biological characteristics of A549 / PEM are different from those of the parental strain. The generation of pemetrexed secondary resistance in lung adenocarcinoma may be related to the up-regulation of HER2 / neu expression.