非小细胞肺癌HCC827奥希替尼耐药细胞株的建立及耐药机制探讨

Establishment of non-small cell lung cancer HCC827 Osimertinib-resistant cell line and exploration of the resistance mechanism

目的体外构建人非小细胞肺癌(NSCLC)奥希替尼耐药细胞株HCC827OR,并探讨耐药机制。方法使用NSCLC细胞株HCC827,通过浓度递增法构建奥希替尼耐药细胞株HCC827OR,并比较两种细胞株形态学、增殖能力、细胞凋亡以及细胞蛋白质表达的差异。结果半抑制浓度(IC50)及耐药指数(RI)结果显示,HCC827OR对奥希替尼高度耐受,且HCC827OR与HCC827细胞形态差异明显。CCK8增殖实验第1天,HCC827OR的细胞活性高于HCC827(P<0.05),第2、3、4天,HCC827OR的细胞活性显著高于HCC827(P<0.01)。克隆形成实验结果显示,HCC827OR集落形成数高于HCC827(P<0.01)。EdU染色后,HCC827OR的EdU阳性细胞数高于HCC827(P<0.01)。凋亡实验结果显示,与HCC827比较,HCC827OR细胞凋亡减少(P<0.01)。Western blot实验结果显示,相较于HCC827,HCC827OR细胞的EGFR和p-EGFR的表达下降,p-AKT、p-ERK和Vimentin的表达上升。结论成功构建NSCLC奥希替尼耐药细胞株HCC827OR,推测耐药机制与EGFR、p-EGFR表达减少,p-AKT、p-ERK和Vimentin表达增加有关。

Objective To construct human non-small cell lung cancer(NSCLC)Osimertinib-resistant cells HCC827OR in vitro,and to explore its resistance mechanism.Methods Using the NSCLC cell line HCC827,the Osimertinib-resistant cell line HCC827OR was constructed by the concentration-increasing method,and the differences in morphology,proliferation,apoptosis,and cell protein expression between the two cell lines were compared.Results Half-inhibitory concentration(IC50)and resistance index(RI)results showed that HCC827OR was highly resistant to Osimertinib,and the morphology of HCC827OR and HCC827 cells were significantly different.On the first day of the CCK8 proliferation experiment,the cell viability of HCC827OR was higher than that of HCC827(P<0.05),and on the second,third and fourth days,the cell viability of HCC827OR was significantly higher than that of HCC827(P<0.01).In the clone formation experiment result showed that,the colony formation number of HCC827OR was higher than that of HCC827(P<0.01).After EdU staining,the number of EdU-positive cells in HCC827OR was higher than that in HCC827(P<0.01).In the apoptosis experiment result showed that,compared with HCC827,the apoptosis of HCC827OR cells was reduced(P<0.01).In Western blot experiment,compared with HCC827,the expression of EGFR and p-EGFR in HCC827OR cells decreased,and the expression of p-AKT,p-ERK and Vimentin increased.Conclusion The NSCLC osimertinib-resistant cell line HCC827OR is successfully constructed.It is speculated that the resistance mechanism is related to the decreased expression of EGFR and p-EGFR,and the increased expression of p-AKT,p-ERK and Vimentin.