CAJ | 학술논문

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.
목적 탐토길비체니획득성내약적비소세포폐암세포주PC9/AB2적상관내약궤제.방법 체외배양인폐선암세포주PC9병채용N-갑기-N'-초기-N-아초기고(MNNG)유변화약물사선건립길비체니획득성내약세포주PC9/AB2,사갑기우담서염(MTF)법검측PC9화PC9/AB2세포대길비체니적민감성,류식세포의검측길비체니대PC9화PC9/AB2세포조망적영향,추제기인조DNA,대상피세포생장인자수체(EGFR)기인19～21외현자진행취합매련반응(PCR)확증급측서,채용Western blot법검측PC9화PC9/AB2세포중c-MET화정합소β1단백적표체수평.세포점부시험화세포화흔시험검측PC9화PC9/AB2세포적점부급천이능력.결과 (1)MTT화조망검측결과표명,PC9/AB2세포주적반수치사농도(IC50)위(24.2±5.5)μmol/L,야생형PC9세포적IC50위(0.04±O.01)μmol/L,여PC9세포상비,PC9/AB2세포대길비체니적내수성제고료576배;재동등제량적길비체니작용하,PC9세포적조망솔위38.5%,이PC9/AB2세포적조망솔위2.2%.(2)기인측서결과증명,PC9화PC9/AB2세포균존재19외현자15 bp적감기결실,단미발생T790M돌변.(3)Western blot검측결과현시,PC9화PC9/AB2세포중적c-MET단백표체수평무명현차이,이정합소β1단백표체수평PC9/AB2세포교PC9세포승고료2.5배.(4)세포점부시험화화흔실험결과표명,PC9/AB2세포적점부화천이능력균명현고우PC9세포.결론 정합소β1수평승고급기상관적종류세포점부、천이능력적제고가능여비소세포폐암PC9/AB2세포주EGFR락안산격매억제제내약상관.
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.