非小细胞肺癌奥希替尼耐药细胞株的建立与鉴定及耐药后对化疗的敏感性

Establishment and identification of an osimertinib-resistant non-small cell lung cancer cell line and sensitivity to chemotherapy after drug resistance

目的:建立非小细胞肺癌奥希替尼耐药细胞株H1975AR,对亲本和耐药细胞株进行鉴定,探讨耐药机制。方法:在体外采用浓度递增的方法予奥希替尼处理诱导H1975细胞建立奥希替尼耐药细胞株H1975AR;光镜及Giemsa染色比较2种细胞形态学差异;第二代测序(next generation sequencing,NGS)检测表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变;磺酰罗丹明B(sulforthodamine B,SRB)法检测细胞增殖,评估细胞对EGFR-酪氨酸激酶抑制剂(tyrosine kinase inhibitor,TKI)和化疗药的敏感性;分别予0、10、100、1000 nmol/L奥希替尼处理6 h,Western blot法检测EGFR及下游增殖信号的变化。结果:①SRB法测得H1975AR的耐药指数为3634.75,显示H1975AR是奥希替尼耐药细胞株;②耐药细胞株形态发生改变且核浆比增大;③与H1975相比,给予奥希替尼处理对H1975AR的p-EGFR和p-ERK抑制作用不明显,且相同的给药浓度对H1975AR的p-AKT抑制作用较弱,同时下调c-Met的表达;④H1975和H1975AR对第一代EGFR-TKI耐药,对顺铂和紫杉醇敏感。结论:H1975AR细胞EGFR C797S顺式突变的产生是奥希替尼主要耐药机制,并对顺铂和紫杉醇敏感。

Objective:To establish an osimertinib-resistant non-small cell lung cancer cell line H1975 AR,identify parental and drugresistant cells,and explore the mechanism of drug resistance.Methods:Induction of H1975 by increasing the concentration of osimertinib in vitro was performed to establish osimertinib-resistant cell line H1975 AR.The morphological differences between the two cells were compared by light microscopy and Giemsa staining.Next generation sequencing(NGS)was used to detect epidermal growth factor receptor(EGFR)mutations.Cell proliferation was determined by SRB assay to access sensitivity to EGFR-tyrosine kinase inhibitor(TKI)and chemotherapeutic drugs.Western blot detected the changes of EGFR and downstream proliferation signaling pathway after treating with 0、10、100、1000 nmol/L osimertinib for 6 h.Results:①The resistance index of H1975 AR measured by SRB assay was 3634.75,which indicated that H1975 AR was an osimertinib-resistant cell line.②The morphology of drug-resistant cell line changed and the nuclear-to-cytoplasmic ratio increased.③Compared with H1975,the inhibitory effected on p-EGFR and p-ERK in H1975 AR were not obvious,and the same dose concentration had a weak inhibitory effect on p-AKT in H1975 AR,and the expression of c-Met was down-regulated.④H1975 and H1975 AR were resistant to the first-generation EGFR-TKI and sensitive to cisplatin and paclitaxel.Conclusion:The EGFR mutation of cis-C797 S in H1975 AR cells is the main mechanism of osimertinib resistance and H1975 AR is sensitive to cisplatin and paclitaxel.