吉非替尼获得性耐药的非小细胞肺癌细胞株PC9／AB2相关耐药机制研究  

Drug resistance mechanism of non small cell lung cancer PC9/AB2 cell line with acquired drug resistance to gefitinib

作　　者：鞠立霞[1] 周彩存[1] 唐亮[1] 赵印敏[1] 杨晓君[1] 粟波[1] 孟淑燕[1] 李玮[1] 严令华[1] 丁永梅[1] 

机构地区：[1]同济大学附属上海市肺科医院肿瘤科,200433

出　　处：《中华结核和呼吸杂志》2010年第5期354-358,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：基金项目：国家A然科学基金资助项目（30873023）;上海市科委课题资助项目（06DZ19502）

摘　　要：目的 探讨吉非替尼获得性耐药的非小细胞肺癌细胞株PC9/AB2的相关耐药机制.方法 体外培养人肺腺癌细胞株PC9并采用N-甲基-N＇-硝基-N-亚硝基胍（MNNG）诱变和药物筛选建立吉非替尼获得性耐药细胞株PC9/AB2,四甲基偶氮唑盐（MTF）法检测PC9和PC9/AB2细胞对吉非替尼的敏感性,流式细胞仪检测吉非替尼对PC9和PC9/AB2细胞凋亡的影响,抽提基因组DNA,对上皮细胞生长因子受体（EGFR）基因19～21外显子进行聚合酶链反应（PCR）扩增及测序,采用Western blot法检测PC9和PC9/AB2细胞中c-MET和整合素β1蛋白的表达水平.细胞黏附试验和细胞划痕试验检测PC9和PC9/AB2细胞的黏附及迁移能力.结果 （1）MTT和凋亡检测结果表明,PC9/AB2细胞株的半数致死浓度（IC50）为（24.2±5.5）μmol/L,野生型PC9细胞的IC50为（0.04±O.01）μmol/L,与PC9细胞相比,PC9/AB2细胞对吉非替尼的耐受性提高了576倍;在同等剂量的吉非替尼作用下,PC9细胞的凋亡率为38.5%,而PC9/AB2细胞的凋亡率为2.2%.（2）基因测序结果证明,PC9和PC9/AB2细胞均存在19外显子15 bp的碱基缺失,但未发生T790M突变.（3）Western blot检测结果显示,PC9和PC9/AB2细胞中的c-MET蛋白表达水平无明显差异,而整合素β1蛋白表达水平PC9/AB2细胞较PC9细胞升高了2.5倍.（4）细胞黏附试验和划痕实验结果表明,PC9/AB2细胞的黏附和迁移能力均明显高于PC9细胞.结论 整合素β1水平升高及其相关的肿瘤细胞黏附、迁移能力的提高可能与非小细胞肺癌PC9/AB2细胞株EGFR酪氨酸激酶抑制剂耐药相关.Objective To study the drug resistance mechanism of non-small cell lung cancer （NSCLC） cell line PC9/AB2 with acquired drug resistance to gefitinib. Methods The human lung adenocarcinoma cell line PC9 was cultured in vitro, and was induced by MNNG to obtain the cell line PC9/AB2 with acquired drug resistance to gefitinib. The sensitivity of the cell line PC9 and PC9/AB2 to gefitinib was determined by MTT assay. The effects of gefitinib on cell apoptosis of the 2 cell lines were determined by flow cytometry. The genomic DNA of the 2 cell lines were extracted, and then the exons 19 -21 of EGFR gene were amplified by PCR and sequenced. The protein expression of c-MET and integrin β1 in the 2 cell lines was determined by Western blot method. The adhesion ability and migration ability of the 2 cell lines were determined by adhesion test and scratch assay. Results （1） The data form MTT and apoptosis detection showed that the IC5o of PC9/AB2 cells was （24.2±5.5） μmol/L,576 times higher than PC9 cells [IC50（0.04±0.01）μmol/L]. Given the same concentration of gefitinib, the apoptosis rate of PC9 cells was 38.48%, while that of PC9/AB2 cells was 2. 2%. （2） The results of gene sequencing showed that there was a deletion of 15 bp in both exon 19 of the 2 cell lines, while no T790M mutation occurred. （3）The results from Western blot showed that there was no significant difference in protein expression of c-MET between the 2 cell lines, while the protein expression of integrin β1 in PC9/AB2 cells was significantlyhigher than that of the PC9 cells. （4） The result from adhesion test and scratch assay showed that the adhesion ability and migration ability of the PC9/AB2 cells was significantly higher that those of PC9 cells.Conclusion The high expression of integrin β1 may be associated with acquired drug resistance of NSCLC cell line PC9/AB2 to gefitinib.

关 键 词：癌 非小细胞肺 抗药性 肿瘤 

分 类 号：R734.2[医药卫生—肿瘤]