X线照射对NSCLC细胞株吉非替尼获得性耐药的体外逆转作用观察  

Reversal of gefitinib-acquired resistance in non-small-cell lung cancer in vitro by X-ray

作　　者：程燕君 谢波[2] 张为民[1,2] CHENG Yanjun;XIE bo;ZHANG Weimin(Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东药科大学,广州510006 [2]广州军区广州总医院

出　　处：《山东医药》2018年第23期1-4,共4页Shandong Medical Journal

基　　金：国家自然科学基金项目(81472172)

摘　　要：目的观察X线照射对非小细胞肺癌(NSCLC)细胞株吉非替尼获得性耐药的体外逆转作用。方法选取表皮生长因子受体(EGFR)基因第19外显子突变的NSCLC细胞株PC-9为对照组,将吉非替尼诱导产生获得性耐药的NSCLC细胞株PC-9/AB分为耐药组和实验组,其中实验组细胞为耐药组细胞经放射线照射(DT:24 Gy/6Gy/4 F)后建立的放射线照射细胞株PC-9/AB IR。各组细胞分别给予不同浓度的吉非替尼,测算吉非替尼对各组细胞生长的半数抑制浓度(IC50)值以评价细胞对吉非替尼的敏感性。采用克隆成型实验观察各组细胞的放射敏感性。通过二代测序法(NGS)检测295基因panel观察各组细胞基因突变情况。采用Western-Blotting法检测各组细胞中的E-cadherin、Vimentin蛋白。采用流式细胞术测算各组细胞的凋亡率。结果吉非替尼抑制对照组、耐药组、实验组细胞生长的IC50值分别为(0.05±0.03)、(16.01±3.42)、(0.28±0.01)μmol/L,其中实验组IC_(50)值低于耐药组(P<0.05)。克隆成型实验结果显示,对照组、耐药组、实验组细胞的存活分数分别为0.579±0.044、0.688±0.011、0.823±0.009,实验组细胞存活分数高于耐药组(P<0.05)。NGS测序结果显示实验组、耐药组均为EGFR基因第19外显子缺失突变细胞,且均携带T790M二次突变,均有TP53基因第7外显子的缺失突变。耐药组细胞中E-cadherin相对表达量低于对照组,实验组E-cadherin相对表达量高于耐药组(P均<0.05);耐药组细胞中vimentin相对表达量高于对照组,实验组vimentin相对表达量低于耐药组(P均<0.05)。20、40、80μmol/L吉非替尼作用后,实验组细胞凋亡率均高于耐药组(P均<0.05)。结论 X线照射可逆转NSCLC细胞株对吉非替尼的获得性耐药,增强细胞对吉非替尼的敏感性,其机制可能与逆转EMT及促进细胞凋亡有关。Objective To explore the reverse effect of ionizing radiation on gefitinib-induced acquired resistance in non-small-cell lung cancer（ NSCLC） cell lines in vitro. Methods The epidermal growth factor receptor（ EGFR） exon19 gene mutation NSCLC cell line PC-9（ control group） and the acquired gefitinib-resistant NSCLC cell line PC-9/AB（ gefitinib-resistant group） were used in this study. PC-9/AB cells were irradiated to induce the radiation-resistant cells,named as PC-9/AB IR（ experimental group）. Different concentrations of gefitinib were given to cells of each group and IC_（50） values of each group were measured to evaluate cell sensitivity to gefitinib. Radiosensitivity was determined by clone forming assay. EGFR gene mutation was detected by the next generation sequencing assay（ 295 gene panel）. The expression of E-cadherin and Vimentin was detected by Western blotting. The apoptosis rate was detected by flow cytometry. Results The IC50 values of the control group,gefitinib-resistant group,and experimental group were（ 0. 05 ± 0. 03）,（ 16. 01 ± 3. 42） and（ 0. 28 ± 0. 01） μmol/L,respectively,and IC50 value of the experimental group was lower than that of the gefitinib-resistant group（ P〈0. 05）. Clone forming assay showed that the cell surviving fraction of the control group,gefitinib-resistant group,and experimental group were 0. 579 ± 0. 044,0. 688 ± 0. 011,and 0. 823 ± 0. 009,respectively;the cell surviving fraction of the experimental group was higher than that of the gefitinib-resistant group（ P〈0. 05）. The next generation sequencing assay showed that gefitinib-resistant group and experimental group harbored the deletion mutation in the exon 19 with T790 M secondary mutation in exon 20 of EGFR and deletion mutation in the exon 7 of TP53. The relative expression of E-cadherin was lower in the gefitinib-resistant group than in the control group,higher in the experimental group than in the gefitinib-resistant group（ both P〈0. 05）. The relative express

关 键 词：X射线 非小细胞肺癌 表皮生长因子受体酪氨酸激酶抑制剂 吉非替尼 获得性耐药 

分 类 号：R734.2[医药卫生—肿瘤]