稳定表达ABCG2细胞模型的建立及其在筛选ABCG2抑制剂中的应用  

Establishment of a Cell Model Stably Expressing ABCG2 and Its Application in Screening Inhibitors of ABCG2

作　　者：郑凤欣 赵泽安 林雪曼 吴婷[1] 庞建新[1] ZHENG Feng-xin;ZHAO Ze-an;LIN Xue-man;WU Ting;PANG Jian-xin(School of Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,Guangdong,China)

机构地区：[1]南方医科大学药学院,中国广东广州510515

出　　处：《生命科学研究》2022年第2期117-124,共8页Life Science Research

基　　金：国家自然科学基金资助项目(81974507,82003819)。

摘　　要：本研究构建了一种稳定表达ATP结合盒转运蛋白2(ATPbinding cassette subfamilyGmember 2,ABCG2)并可用于筛选ABCG2抑制剂的细胞模型。首先,利用脂质体lipo3000将表达载体pcDNA3.1(+)-ABCG2转染至HEK293细胞,经G418筛选阳性克隆株后,采用qRT-PCR与Western-blot方法进行ABCG2稳转细胞株(稳转株)的鉴定;随后,利用差速离心法提取膜囊泡,通过14C-尿酸同位素摄取实验筛选ABCG2抑制剂。结果表明,ABCG2稳转株的mRNA及蛋白质表达水平分别为野生型的1717.5倍和2.64倍;经其提取的囊泡摄取14C-尿酸的能力为对照囊泡的3.73倍。利用上述工具细胞,研究评价了临床常用的降尿酸药物对ABCG2的抑制活性。结果显示,苯溴马隆抑制ABCG2的IC50值为(3.45±1.09)μmol/L,RDEA3170抑制ABCG2的IC50为(9.90±2.11)μmol/L。综上所述,本实验成功构建了ABCG2抑制剂体外筛选模型,并首次发现RDEA3170具有ABCG2抑制作用。To screen ATP binding cassette subfamily G member 2(ABCG2)inhibitors,a stable ABCG2 expression cell model was constructed.The expression vector pcDNA3.1(+)-ABCG2 was transfected into HEK293 cells using lipo3000,and the positive clones were selected by G418.The mRNA and protein expression levels in stable ABCG2 transgenic cells were validated by qRT-PCR and Western-blot methods.Membrane vesicles were then extracted by differential centrifugation and potential ABCG2 inhibitors were screened through 14C-uric acid uptake assay.The results showed that the mRNA and protein levels in ABCG2-expressing cells were 1717.5 and 2.64 times as much as those in mock-transfected cells,respectively.The uric acid uptake activity of the extracted vesicles from ABCG2-expressing cells was 3.73 times as much as that of control vesicles.In addition,the inhibitory effects of commonly used uric acid-lowering drugs on ABCG2 were evaluated using the obtained cell model.The results showed that benzbromarone inhibited ABCG2 with an IC50 value of(3.45±1.09)μmol/L,and RDEA3170 with an IC50 value of(9.90±2.11)μmol/L.In conclusion,the experiments showed successful construction of the screening model of ABCG2 inhibitors and also demonstrated for the first time the potential ABCG2 inhibition capability of RDEA3170.

关 键 词：高尿酸血症(HUA) 转运体 ATP结合盒转运蛋白2(ABCG2) 维立诺雷(RDEA3170) 

分 类 号：Q95-336[生物学—动物学] R965.1[医药卫生—药理学]