目的：构建厄洛替尼耐药人肺腺癌细胞模型PC-9/ER，观察单用人表皮生长因子受体(epithelial growth factor receptor，EGFR)酪氨酸激酶抑制剂厄洛替尼或联合胰岛素样生长因子受体1 (insulin-like growth factor receptor 1，IGF1R)酪氨酸激酶抑制剂苦鬼臼毒(picropodophyllotoxin，PPP)作用于该细胞后，该细胞对厄洛替尼耐药性的影响，并探讨耐药相关机制。方法：选择人肺腺癌细胞株PC-9，采用逐步递增厄洛替尼浓度的方法体外诱导构建耐药株PC-9/ER。CCK-8法检测耐药指数；细胞计数法绘制PC-9和PC-9/ER的生长曲线，并计算出两细胞系的倍增时间；流式细胞术检测两细胞系的细胞周期；Western Blot法检测p-EGFR及p-IGF1R的表达水平，并进一步检测厄洛替尼及PPP单独或联合作用于PC-9/ER后，各组Akt，ERK，p-Akt及p-ERK的表达水平。结果：PC-9/ER细胞株的耐药指数是72.3。细胞生长曲线显示，PC-9/ER细胞生长较亲代细胞慢，PC-9与PC-9/ER细胞的倍增时间分别为32.9及36.9 h (P=0.003)。与PC-9相比，PC-9/ER细胞的G1期细胞增多(P=0.001)，而S期细胞则明显下降(P=0.015)。Western Blot结果表明，PC-9/ER细胞中p-IGF1R表达比亲代细胞明显增多(P=0.016)，而p-EGFR无明显变化(P=0.152)。在亲代及耐药细胞系，厄洛替尼联合PPP组均较其他组更能显著的抑制细胞增殖(P<0.05)；且Western Blot法表明联合用药组EGFR下游磷酸化的Akt、ERK的表达水平明显减少。结论：成功构建了人肺腺癌厄洛替尼耐药细胞株PC-9/ER，IGF1R通路可能与肺腺癌EGFR-TKI获得性耐药有关。
Establishment and characterization of a erlotinib—drug resistant variant of human lung adenocarcinoma cell line PC-9/ER
Objective: To establish a human lung adenocarcinoma cell line PC-9/ER that is resistant to erlotinib; to observe the effect of erlotinib alone or in combination with PPP (a tyrosine kinase inhibitor of insulin-like growth factor receptor 1) on PC-9/ER and to discuss its possible mechanism. Methods: A erlotinib resistant human lung adenocarcinoma cell line PC-9/ER was induced by continuously exposing human lung adenocarcinoma cell line PC-9 to gradually increased doses of erlotinib. The drug resistant ability to erlotinib was measured by CCK-8 assay. The growth curve and cell cycle of PC-9 and PC-9/ER were compared. The expression of Akt, ERK, p-EGFR, p-IGF1R, p-Akt and p-ERK was measured by Western Blot in different groups. Results: The drug resistant index of PC-9/ER to erlotinib was 72.3. Double time of PC-9 and PC-9/ER were 32.9 and 36.9 h (P=0.003), respectively, as evaluated by the growth curve. Figures in G1 phase was decreased in PC-9 than PC-9/ER (P=0.001). The expression of p-IGF1R significantly increased in PC-9/ER (P=0.016) but not p-EGFR (P=0.152). More significant inhibition of cell proliferation than other groups was observed in combination group that erlotinib combination with PPP (P<0.05). The expression of p-Akt and p-ERK was decreased in combination group. Conclusion: The resistant cell model PC-9/ER is established and IGF1R may associate with drug resistance to EGFR-TKI.