Gefitinib获得性耐药产生时非小细胞肺癌上皮性标志EGFR及E-cadherin表达的变化

Expressions of EGFR and E-cadherin in non-small cell lung cancer with acquired resistance to gefitinib

[目的]了解gefitinib获得性耐药产生时非小细胞肺癌上皮性标志EGFR及E-cadherin表达的变化。[方法]采用逐步递增药物浓度、间歇作用体外诱导法诱导人肺腺癌NCI-H1975细胞株gefitinib获得性耐药;倒置显微镜下观察亲本细胞株和耐药细胞株在gefitinib作用前后的细胞形态学变化差异;细胞计数法描绘耐药细胞株和亲本细胞株的生长曲线并计算群体倍增时间;免疫细胞化学法检测耐药细胞株及亲本细胞株的EGFR及E-cadherin蛋白表达状态,并比较二株细胞间两种蛋白表达的差异。[结果]历时6个月诱导建成人肺腺癌gefitinib耐药细胞株NCI-H1975/GR。Gefitinib作用后耐药细胞株NCI-H1975/GR的细胞形态变化与亲本株NCI-H1975有所不同;在gefitinib的作用下,NCI-H1975/GR细胞体积变小,部分细胞变为纺锤形;NCI-H1975/GR细胞株群体倍增时间延长7.14 h(P<0.05);免疫细胞化学结果显示:与亲本株比较,虽然耐药细胞株NCI-H1975/GR细胞细胞膜EGFR蛋白的表达无明显变化,但在细胞浆的表达明显增高;E-cadherin在细胞膜的表达明显降低。[结论]非小细胞肺癌产生gefitinib获得性耐药后EGFR蛋白在细胞浆积聚;而以E-cadherin为代表的上皮分化标记表达减弱。

[ Objective] To understand the expression changes of EGFR and E -cadherin as epithelial markers in NSCLC with acquired resistance to gefitinib. [ Methods] The cell line NCI- H1975 was induced to develop gefitinib acquired re- sistance by repeated intermittent exposure to the drug in increasing concentration. The morphological changes between par- ent and drug resistant cell lines before and after gefitinib exposure were observed under inverted microscope. The growth curves of parent and drug resistant cell lines were depicted with eytometry, and population doubling times of parent and drug cell lines were calculated ; The protein expressions of EGFR and E - cadherin were detected by immunocytochemistry and the expression differences of them between parent and drug cell lines were compared. [ Results] The drug resistant cell line NCI - H1975/GR was established for over 6 month. The morphologic change of NCI - H1975/GR cells was different from that of NCI - H1975 cells following exposure to gefitinb. The NCI - H1975/GR cells became smaller and some cells were spindle in shape following exposure to gefitinb. The population doubling time was postponed 7.14 h （ P 〈 0.05 ）. The cytoplasm expression of EGFR of NCI - H1975/GR ceils was significantly stronger than that of NCI - H1975, while no dis- tinct difference of the expression in cell membrane between the two cell lines. The membrane expression of E - cadherin in NCI -H1975/GR cells was weaker than that in NCI - H1975. [ Conclusion] There is EGFR cytoplasm accumulation in NSCLC cells following development of acquired resistance to gefitinib in vitro, but the expression of E - cadherin as the epi- thelial differentiated marker decreases in the ceils.