论文部分内容阅读
目的:探讨EGFR基因启动子甲基化水平与人非小细胞肺癌细胞株H1650对吉非替尼敏感性之间的关系。方法:5-aza-CdR和吉非替尼单独或联合用药作用于H1650细胞株后,应用甲基化特异性PCR法检测EGFR基因启动子区甲基化状态;CCK-8法检测细胞增殖率;流式细胞仪技术检测细胞凋亡率变化;蛋白质印迹法和实时荧光定量PCR法检测EGFR蛋白和mRNA的表达情况。结果:5-aza-CdR可去除H1650细胞EGFR基因启动子区甲基化;CCK-8法检测结果显示,H1650对吉非替尼的敏感性较差,5-aza-CdR去甲基化后联合运用吉非替尼处理72h,联合用药组IC50为(2.93±0.95)μmol/L,与吉非替尼组(14.53±1.13)μmol/L、5-aza-CdR组(4.91±1.42)μmol/L比较显著降低,P<0.05;流式细胞仪技术结果显示,联合用药组细胞凋亡率为(83.62±4.3)%,与吉非替尼组(20.29±2.9)%、5-aza-CdR组(25.73±7.5)%比较,差异有统计学意义,P<0.05;蛋白质印迹法和实时荧光定量PCR法结果显示,吉非替尼与5-aza-CdR联合用药干预72h后细胞EGFR蛋白和mRNA表达显著下降,与单药组相比,具有统计学意义,P<0.05。结论:EGFR基因启动子区甲基化可能是非小细胞肺癌细胞株H1650对吉非替尼获得性耐药的机制之一。
Objective: To investigate the relationship between the promoter methylation of EGFR gene and the sensitivity of human non-small cell lung cancer cell line H1650 to gefitinib. Methods: Methylation-specific PCR was used to detect the methylation status of EGFR gene promoter after 5-aza-CdR and gefitinib treatment alone or in combination with H1650 cell line. CCK-8 assay was used to detect cell proliferation rate The changes of apoptosis rate were detected by flow cytometry. The expression of EGFR protein and mRNA was detected by Western blotting and real-time fluorescence quantitative PCR. Results: 5-aza-CdR could remove the promoter methylation of EGFR gene in H1650 cells. The CCK-8 assay showed that the sensitivity of H1650 to gefitinib was poor. After 5-aza-CdR demethylation The combination therapy with gefitinib for 72h showed IC50 of (2.93 ± 0.95) μmol / L in combination group was significantly higher than that in gefitinib group (14.53 ± 1.13μmol / L, 5-aza-CdR group, 4.91 ± 1.42μmol / (P <0.05). The results of flow cytometry showed that the apoptosis rate of the combination group was (83.62 ± 4.3)%, which was significantly higher than that of the gefitinib group (20.29 ± 2.9)%, 5-aza- CdR group (25.73 ± 7.5)%, the difference was statistically significant, P <0.05; Western blotting and real-time fluorescence quantitative PCR results showed that gefitinib and 5-aza-CdR intervention 72h after the cell EGFR protein And mRNA expression decreased significantly, compared with the single drug group, with statistical significance, P <0.05. Conclusion: The promoter methylation of EGFR gene may be one of the mechanisms of acquired resistance to gefitinib in non-small cell lung cancer cell line H1650.