摘要
目的构建人肾脏近曲小管阴离子转运体1(human organic anion transporter 1,hOAT1)-增强型绿色荧光蛋白(EGFP)质粒,获得稳定表达pEGFP-hOAT1的HEK-293细胞系。方法构建EGFP与hOAT1的融合基因表达载体pEGFP-hOAT1,并利用FuGENE 6转染试剂将其转染至HEK293细胞中,用荧光显微镜观察绿色荧光蛋白表达情况,经G418抗性筛选获得稳定转染细胞株。用RT-PCR法、qRT-PCR法和Western blot技术,检测稳定转染细胞及正常细胞hOAT1 mRNA和蛋白的表达情况,并进一步用超高效液相色谱-串联质谱法(UPLC-MS/MS)考察稳定转染细胞的对氨基马尿酸摄取能力,以及丙磺舒对hOAT1的抑制作用。结果使用该文的方法,细胞转染率可达90%。RT-PCR、qRT-PCR、Western blot,以及对氨基马尿酸摄取、丙磺舒抑制实验结果显示,相对于正常组,转染细胞在荧光显微镜下呈绿色荧光,其hOAT1表达均呈阳性(P<0.01);稳定转染细胞的对氨基马尿酸摄取能力明显升高(P<0.05),且丙磺舒对hOAT1有明显的抑制作用(P<0.05)。结论高效快速地建立了稳定高表达hOAT1的HEK293细胞系,为核苷类膦酸类似物这类药物引起的临床剂量依赖性肾毒性的机制研究奠定了模型基础,也为体外研究hOAT1底物或抑制剂的筛选提供了一个便利的工具。
To construct the human organic anion transporter 1(hOAT1)-enhanced green fluorescent protein(EGFP)plasmid and obtain HEK-293 cell line stably expressing pEGFP-hOAT1.Methods The recombinant plasmid pEGFP-hOAT1 expressing EGFP-hOAT1 fusion protein was constructed,and the recombinant plasmid was transfected into HEK293 cells using FuGENE 6 transfection reagent.Fluorescent microscope was used to observe the expression of green fluorescent protein(GFP).The monoclonal cell lines were selected by G418 resistance screening and expanded to obtain stable transfected cell lines.The expression of hOAT1 mRNA and protein in stable transfected cells and normal cells was detected by RT-PCR,qRT-PCR and Western blot,and p-aminohippuric acid uptake capacity,and probenecid hOAT1 inhibitory effect in stable transfected HEK293 cells were further investigated using UPLC-MS/MS technique.Results Using this method,the cell transfection rate could reach 90%.The results of RT-PCR,qRT-PCR,Western blot and p-aminohippuric acid uptake and probenecid inhibition showed that the hOAT1 expression was positive in the transfected cells under the fluorescence microscope as compared with normal group(P<0.01),the uptake of p-aminohippuric acid in transfected cells significantly increased(P<0.05),and hOAT1 was markedly inhibited by probenecid(P<0.05).Conclusions HEK293 cell line with stable and high expression of hOAT1 has been established efficiently and rapidly,which lays the foundation for the study of the mechanism of clinical dose-dependent nephrotoxicity induced by antiviral nucleoside or nucleotide analogues and also provides a convenient tool for in vitro screening hOAT1 substrates or inhibitors.
作者
李静
杨洋
肖涛
李韬
欧瑜
傅晓钟
刘亭
LI Jing;YANG Yang;XIAO Tao;LI Tao;OU Yu;FU Xiao-zhong;LIU Ting(Guizhou Provincial Key Lab of Pharmaceutics/State Key Lab of Functions and Applications of Medicinal Plant,Guizhou Medical University,Guiyang 550004,China;School of Pharmacy,Guizhou Medical University,Guiyang 550004,China;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM(Ministry of Education),Guizhou Medical University,Guiyang 550004,China;Pharmacy Dept, Maternal and Child Health Hospital of Guiyang Province,Guiyang 550002,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2018年第10期1471-1476,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81460523)
贵州省高等学校创新团队靶向药物研究与开发创新团队(黔教合人才团队字[2015]57号)
贵阳市科技计划项目(筑科合同[2018]1-36号)
