基于细胞模型的抗HER2单抗耐药通路研究方法的建立及应用  

Establishment and application of resistance pathway research method of anti⁃HER2 monoclonal antibody based on cellular model

作　　者：王馨 池文杰 WANG Xin;CHI Wenjie(Fujian Institute for Food and Drug Quality Control,Fuzhou 350001,China)

机构地区：[1]福建省食品药品质量检验研究院,福州350001

出　　处：《山西医科大学学报》2024年第11期1449-1455,共7页Journal of Shanxi Medical University

摘　　要：目的 建立基于细胞模型的抗人类表皮生长因子受体2(HER2)单抗耐药通路研究方法。方法 从低剂量开始分别使用原研药曲妥珠(Trastuzumab)或其生物类似药候选物对HER2阳性BT474细胞进行加压筛选至药物浓度60μg/mL。利用流式细胞术检测抗HER2单抗耐药细胞株表面HER2表达量变化。利用细胞增殖抑制生物学活性测定法对加压细胞株进行耐药性验证。对耐药细胞株和敏感细胞株进行高通量转录组测序以挖掘差异表达基因(DEGs)。利用KEGG数据库对得到的DEGs进行通路富集分析,寻找耐药相关通路。结果 经过相同条件的药物加压筛选,原研药及其生物类似药候选物均不改变BT474细胞表面HER2抗原表达量。细胞增殖抑制活性实验表明两种药物的加压细胞株均呈现一定耐药性,且耐药曲线相似。转录组测序结果经过KEGG富集分析表明,与敏感细胞相比,原研药和生物类似药候选物的耐药细胞株的DEGs均在丝裂原活化蛋白激酶通路及神经活性的配体-受体相互作用通路有富集。不同的是,原研药耐药细胞株的DEGs还在黏着斑激酶通路及肌动蛋白细胞骨架调节通路上有富集,而生物类似药候选物耐药细胞株则在细胞因子-细胞因子受体相互作用通路上有富集。结论 建立了基于细胞模型的抗HER2单抗耐药通路研究方法,可用于比较原研药曲妥珠与其生物类似药候选物两者的耐药相关基因通路的异同,为单抗生物类似药的耐药研究提供参考。Objective To establish a method for studying the resistance pathway of anti-HER2 monoclonal antibodies based on the cellular model.Methods Starting from low doses,HER2 positive BT474 cells were respectively subjected to pressure using the original drug Trastuzumab or its biosimilar drug candidate at a drug concentration of 60µg/mL.The changes of HER2 expression on the surface of anti-HER2 monoclonal antibody resistant cell lines were detected by flow cytometry.Cell proliferation inhibition assay was conducted to validate the drug-resistance of cells.High-throughput transcriptome sequencing was performed on resistant and sensi-tive cell lines to identify differentially expressed genes(DEGs).Then the identified DEGs were conducted by enrichment analysis of KEGG signaling pathways to identify the pathways related to resistance.Results After drug pressure screening under the same con-ditions,the expression of HER2 antigen on BT474 was not changed by either Trastuzumab or its biosimilar candidate.The results of cell proliferation inhibition assay showed the cells subjected to two drugs both had certain resistance,and the resistant curves were similar.KEGG enrichment analysis of transcriptome sequencing showed that the DEGs between the resistant cell line and the sensitive cells of the reference drug and its biosimilar drug candidates were enriched in the mitogen-activated protein kinase pathway and neuro-active ligand-receptor interaction pathway.Differently,the DEGs in the resistant cell line of the reference drug were also enriched in the focal adhesion kinase pathway and regulation of actin cytoskeletion pathway,while the DEGs in resistant cell line of the biosimilar drug candidates were enriched in cytokine-cytokine receptor interaction pathway.Conclusion A method based on cellular model is successfully established to study the resistance pathway of anti-HER2 monoclonal antibodies,and can be used to compare the resis-tance-related gene pathways of the original drug Trastuzumab with its biosimilar candidate drugs,

关 键 词：曲妥珠单抗 生物类似药 耐药细胞 转录组测序 差异表达基因 丝裂原活化蛋白激酶通路 神经活性的配体-受体相互作用通路 

分 类 号：R92[医药卫生—药学]