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目的培美曲塞是治疗非鳞癌非小细胞肺癌(non-small cell lung cancer,NSCLC)的常用药物,其对表皮生长因子受体(pithelial growth factor receptor,EGFR)敏感突变的肺腺癌是否具有放射增敏作用尚未明确。本课题研究培美曲塞在体外对EGFR 19外显子突变的人肺腺癌细胞株PC9的放射增敏作用,并初步探讨其作用机制。方法以PC9细胞作为研究对象,采用MTT法检测培美曲塞的20%抑制浓度(20%inhibition concentration,IC_(20)),将PC9细胞分为对照组、单独照射组、培美曲塞单药组和培美曲塞+照射组4组;采用流式细胞术检测培美曲塞联合或不联合照射对PC9细胞的凋亡率及细胞周期的影响;克隆形成实验检测培美曲塞对PC9细胞的放射效应,计算存活分数,拟合存活曲线;蛋白质印迹法检测CDC20蛋白在各组的表达。结果 MTT结果显示,PC9细胞的增殖抑制与培美曲塞呈时间-剂量依赖性,取48h的IC_(20)(0.084μmol/L)的培美曲塞作为实验浓度。流式细胞术结果显示,培美曲塞单药组和单独照射组均可增加凋亡率,分别为(7.17±1.14)%和(10.78±1.52)%,而培美曲塞+照射组具有协同增效作用,凋亡率达(29.23±1.33)%,F=227.57,P<0.001;细胞周期结果显示,对照组G_2/M期比例为(0.79±0.63)%,培美曲塞单药组为(0.79±0.47)%,单独照射组为(18.21±0.72)%,培美曲塞+照射组为(25.09±1.04)%,提示培美曲塞使细胞阻滞在S期,与放射线联合作用后,S期细胞比例减少,G_2/M期的比例明显增多,差异有统计学意义,F=276.85,P<0.001。克隆形成实验提示,培美曲塞具有较好的放射增敏作用,其放射增敏比(sensitization enhancement ratio,SER)为1.41。培美曲塞联合照射能增加细胞周期相关蛋白CDC20的表达,F=282.12,P<0.001。结论培美曲塞可提高EGFR 19外显子突变的PC9肺腺癌细胞的放射敏感性,其机制可能与照射引起CDC20的增加致对放射敏感的G_2/M期阻滞,而照射耐受的S期比例减少有关。
OBJECTIVE: Pemetrexed is a commonly used drug for the treatment of non-small cell lung cancer (NSCLC), and whether it has an effect on lung adenocarcinoma with sensitive mutations of EGFR (EGFR) Radiosensitization has not yet been clarified. This topic pemetrexed in vitro EGFR 19 exon mutations in human lung adenocarcinoma cell line PC9 radiosensitization and preliminary study of its mechanism of action. Methods PC9 cells were used as research objects. 20% inhibition concentration (20% inhibition concentration) of pemetrexed was detected by MTT method. PC9 cells were divided into control group, irradiation group, pemetrexed alone Drug group and pemetrexed + irradiation group. Flow cytometry was used to detect the apoptosis rate and cell cycle of PC9 cells treated with or without pemetrexed. The clonogenic assay was used to detect pemetrexed PC9 cells, the survival score was calculated and the survival curve was fitted. The expression of CDC20 protein in each group was detected by Western blotting. Results The MTT results showed that the inhibition of proliferation of PC9 cells was in a dose-dependent manner with pemetrexed, and the concentration of pemetrexed at IC 20 (0.084 μmol / L) 48 h was used as the experimental concentration. The results of flow cytometry showed that the pemetrexed monotherapy group and the irradiation alone group could increase the apoptosis rate (7.17 ± 1.14)% and (10.78 ± 1.52)%, respectively. Pemetrexed + irradiation group had (29.23 ± 1.33)%, F = 227.57, P <0.001. The results of cell cycle showed that the proportion of G_2 / M phase in control group was (0.79 ± 0.63)%, the rate of apoptosis of pemetrexed monotherapy (0.79 ± 0.47)% in group A and (18.21 ± 0.72)% in group B and (25.09 ± 1.04)% in pemetrexed + irradiation group, suggesting that pemetrexed blocks cells in S phase and radiotherapy After combined treatment, the proportion of cells in S phase decreased and the proportion of G 2 / M phase increased significantly, the difference was statistically significant (F = 276.85, P <0.001). Clone formation experiments suggest that pemetrexed has better radiosensitization with a sensitization enhancement ratio (SER) of 1.41. Pemetrexed combined irradiation increased the expression of cell cycle associated protein CDC20, F = 282.12, P <0.001. Conclusion Pemetrexed can increase the radiosensitivity of PC9 lung adenocarcinoma cells with EGFR 19 mutation. The mechanism may be related to the blockage of radiation-sensitive G 2 / M phase induced by the increase of CDC20, while the radiation-tolerant S phase reduction related.