摘要
目的旨在建立核磷蛋白1(nucleophosmin 1,NPM1)稳定敲除的耐顺铂膀胱癌细胞系。方法采用慢病毒感染的方式,对耐顺铂膀胱癌细胞系T24/DDP的NPM1基因进行敲除,并采用RT-PCR(reverse transcription-polymerase chain reaction)和Western blotting法在基因水平和蛋白水平对NPM1的敲除效果进行验证,并通过荧光显微镜验证荧光效率。采用有限稀释法对感染后的细胞进行筛选纯化,通过单克隆增殖的方法对纯化后的细胞增殖培养,建立具有NPM1稳定敲除的耐顺铂膀胱癌细胞系。结果通过基因和蛋白水平比较筛选出能够有效敲除NPM1的基因片段,并通过单克隆筛选获得了NPM1稳定敲除的耐顺铂膀胱癌细胞系。筛选后细胞系荧光效率近100%,且对NPM1有明显敲除效率。结论NPM1稳定敲除的耐顺铂膀胱癌细胞系T24/DDP具有稳定的NPM1基因敲除效果,可以作为研究膀胱癌细胞耐药的良好实验模型。
Objective To establish a cisplatin-resistant bladder transitional cell carcinoma cell line with stable knockout of NPM1.Methods NPM1 gene of Cisplatin-resistant bladder cancer cell line T24/DDP was knocked out by lentivirus infection.The knockout effect of NPM1 was verified by reverse transcription-polymerase chain reaction(RT-PCR)and western blotting,and the fluorescence efficiency was verified by fluorescence microscopy.The infected cells were screened and purified by limited dilution method.The purified cells were then cultured by monoclonal proliferation method to establish a cisplatin-resistant bladder transitional cell carcinoma cell line with stable knockout of NPM1.Results NPM1 gene fragments were effectively knocked out by comparing gene and protein levels,and the stable knockout of NPM1-resistant bladder transitional cell carcinoma cell line was obtained by monoclonal screening.After screening,the fluorescence efficiency of the cell lines was nearly 100%,and the knockout efficiency of NPM1 was robust.Conclusion The stable knockout of NPM1 in cisplatin-resistant bladder cancer cell line T24/DDP has stable knockout effect of NPM1 gene and can be used as a optimal experimental model to study drug resistance of bladder cancer cells.
作者
罗陈烁
赵嫚
雷婷
张曼
LUO Chen-shuo;ZHAO Man;LEI Ting;ZHANG Man(Beijing Shijitan Hospital Affiliated to Capital Medical University,Clinical Laboratory Medicine,Beijing Shijitan Hospital,Peking University Ninth School of Clinical Medicine,Department of Clinical Laboratory,Beijing Key Laboratory of Urinary Cellular Molecular Diagnostics,Beijing,100038,China)
出处
《标记免疫分析与临床》
CAS
2019年第9期1560-1564,共5页
Labeled Immunoassays and Clinical Medicine
基金
国家重点研发计划(编号:2016YFC1000702)
北京市医院管理局“登峰”人才培养计划(编号:DFL20150701)
关键词
NPM1
耐药膀胱癌
顺铂
细胞感染
细胞模型
NPM1
Drug-resistant bladder cancer
Cisplatin
Cell infection
Cell model
