稳定表达EGFR胞外结构域A289V突变的恶性胶质瘤细胞系的构建及鉴定

Construction and Identification of Glioma Cell Lines Stably Expressing EGFR Extracellular Domain A289V Mutation

利用慢病毒转染技术构建两种能够稳定表达EGFR ECD A289V错义点突变的胶质瘤细胞系,为该种突变胶质瘤的临床前研究提供细胞模型。使用同源重组酶连法构建重组质粒,通过钙转法包装慢病毒感染U87、U251胶质瘤细胞,Western blot验证EGFR A289V突变蛋白的表达情况,Transwell侵袭实验观察两种细胞系侵袭能力的变化,并利用CCK-8细胞活力检测实验探究TKI对其的有效性。结果显示,所构建的质粒测序鉴定成功,并且重组质粒感染目的细胞系后可顺利表达EGFR A289V突变蛋白。经Transwell侵袭实验和CCK-8细胞活力检测实验确认改造过的U87、U251细胞系侵袭能力增加,且能被奥希替尼有效抑制。综上,表达EGFR A289V突变的胶质瘤细胞系更具侵袭表现,奥希替尼在体外环境中能够有效抑制存在此类突变的胶质瘤细胞的增殖。该实验为此种突变的药理学研究提供了可供使用的细胞模型。

The aim of this study was to construct two glioma cell lines with stable expression of EGFR ECD A289V missense point mutation by lentivirus transfection technique,and to provide a cell model for the preclinical study of this mutant glioma.The recombinant plasmid was constructed using homologous recombination enzyme linking method and the lentivirus was packaged by calcium transfer method to infect U87 and U251 glioma cells.Verification via Western blot confirmed the expression of EGFR A289V mutant protein,while Transwell invasion assay observed changes in invasion ability of both cell lines.The efficacy of TKI on CCK-8 cell viability was investigated via CCK-8 cell viability assay.Results showed that constructed plasmid was identified successfully by sequencing,and the recombinant plasmid could successfully express EGFR A289V mutant protein after infecting the target cell line.Transwell invasion assay and CCK-8 cell viability assay confirmed that the invasion ability of modified U87 and U251 cell lines was increased and could be effectively inhibited by Osimertinib.Overall,glioma cell lines expressing EGFR A289V mutation are more malignant,and Osimertinib can effectively inhibit the proliferation of glioma cells with such mutation in vitro.This experiment provides a useful cell model for the pharmacological study of this mutation.