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稳定表达TFEB-GFP细胞系的构建及促TFEB入核药物筛选

Construction of Stably Expressed TFEB-GFP Cell Line and Screening of Drugs Promoting TFEB Nuclear Translocation
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摘要 本研究旨在构建稳定表达TFEB-GFP的HeLa细胞系,并利用其筛选促进转录因子EB(TFEB)入核药物.利用分子生物学技术成功构建了重组表达载体pLVX-AcGFP1-N1-TFEB,并进行慢病毒包装,进而侵染HeLa细胞,通过嘌呤霉素筛选阳性单克隆细胞株.采用荧光显微镜技术及免疫印迹技术(Western blot)证实稳定表达TFEB-GFP细胞株构建成功.利用该细胞模型筛选得到促TFEB入核药物盐酸舍曲林(Sertraline HCl),此药物能够促进转录因子TFEB靶基因的表达,并促进细胞自噬.MTT实验表明Sertraline HCl抑制肿瘤细胞活力,其对肿瘤细胞的杀伤作用可能是通过其激活自噬引起的. The purpose of this study is to construct a HeLa cell line which can stably express TFEB-GFP and be used to screen drugs so as to promote TFEB nuclear translocation.The recombinant expression vector,pLVX-AcGFP1-N1-TFEB,was successfully constructed using molecular biology techniques.Encapsulated-lentiviruses were prepared and used to infect HeLa cells.Positive monoclonal cell lines expressing TFEB-GFP were generated after puromycin screening and verified with fluorescence microscopy technique and Western blot.Sertraline HCl,a drug promoting TFEB into nuclei,was screened using this cell model.Sertraline HCl increased the expression of the transcription factor TFEB target genes and promoted cell autophagy.MTT assay showed that Sertraline HCl inhibited cancer cell viability,and this inhibiting effect might be caused by its activation of autophagy.
作者 李薇 刘振兴 杨萌 赵爽 刁爱坡 LI Wei;LIU Zhenxing;YANG Meng;ZHAO Shuang;DIAO Aipo(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;College of Basic Medical,Inner Mongolia Medical University,Hohhot 010110,China)
出处 《天津科技大学学报》 CAS 2019年第5期21-28,34,共9页 Journal of Tianjin University of Science & Technology
基金 国家自然科学基金资助项目(31471335)
关键词 转录因子EB 核转位 盐酸舍曲林 细胞自噬 TFEB nuclear translocation Sertraline HCl autophagy
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