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目的:建立非小细胞肺癌(NSCLC)第三代EGFR-TKI奥希替尼(Osimertinib)耐药细胞系并初步探讨其耐药机制。方法:以人NSCLC细胞系H1975为研究对象，利用低浓度奥希替尼持续诱导筛选继发耐药细胞系。MTS法检测细胞奥希替尼药物敏感性。活细胞工作站检测细胞增殖能力。流式细胞仪检测细胞周期与细胞凋亡。利用蛋白质谱检测数据构建亲代与耐药细胞的差异表达基因谱并通过qRT-PCR和Wester blot验证耐药相关基因。结果:成功获得奥希替尼继发耐药H1975/OSI细胞。与亲代细胞相比，H1975/OSI细胞的耐药指数增加了27.25倍(n P<0.01)，细胞增殖能力降低但凋亡抗性增加(n P＝0.01)且未产生EGFR-TKI耐药常见相关基因的突变。同时建立起H1975与H1975/OSI细胞蛋白质差异表达谱，发现耐药细胞上调蛋白307个，下调蛋白295个，其中在H1975/OSI细胞中表达增加的成纤维细胞特异蛋白(FSP1)被干扰后，干扰细胞对奥希替尼的敏感性增加(IC50值下降3.51倍，n P＝0.02)。在亲代H1975细胞中过表达FSP1，奥希替尼对细胞的IC50值增加了3.75倍(n P<0.01)。n 结论:成功建立人NSCLC奥希替尼耐药细胞系H1975/OSI；成功构建H1975与H1975/OSI蛋白质差异表达谱；FSP1蛋白可能通过增加H1975/OSI的凋亡抗性介导奥希替尼耐药。“,”Objective:To establish osimertinib-resistant non-small cell lung cancer (NSCLC) cell line and explore its drug resistance mechanism.Methods:The human NSCLC cell line H1975 was used as the research object, and low-concentration osimertinib was used to continuously select secondary drug-resistant cell lines. Osimertinib drug sensitivity of cells was detected by MTS method. Cell proliferation was detected by live cell workstations. Flow cytometry was used to detect cell cycle and apoptosis. Protein mass spectrometry was used to construct differentially expressed protein profiles between parental and drug-resistant cells and some resistance-related proteins were validated by real time fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western blot.Results:Secondary drug-resistant H1975/OSI cell line were successfully established. Compared with the parental cells, the resistance index of H1975/OSI cells increased by 27.25 times (n P<0.01), the cell proliferation ability decreased but the apoptosis resistance increased (n P=0.01), and no new drug-resistance related gene mutation in H1975/OSI cells. Meanwhile, the differential protein expression profiles of H1975 and H1975/OSI cells were built, and 307 upregulated proteins and 295 down-regulated proteins were found in resistant cells. When fibroblast specific protein-1 (FSP1) gene with expression up-regulation was diturbed in H1975/OSI cells, the cell IC50 value of osimertinib decreased 3.51 times (n P=0.02) , and when FSP1 was overexpressed in the H1975 cells, the IC50 value of osimertinib increased by 3.75 times (n P<0.01).n Conclusions:We successfully established human NSCLC osimitinib-resistant cell line H1975/OSI. Protein differential expression profiles between H1975 and H1975/OSI was constructed successfully. It was found that FSP1 was involved in mediating the resistance of H1975/OSI to osimertinib.