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CYP3A22稳定表达HepG2细胞株的建立及其探针药物代谢活性鉴定
引用本文:薛正楷,魏泓,商海涛,杨根庆,叶彬. CYP3A22稳定表达HepG2细胞株的建立及其探针药物代谢活性鉴定[J]. 第三军医大学学报, 2009, 31(16): 1552-1555
作者姓名:薛正楷  魏泓  商海涛  杨根庆  叶彬
作者单位:重庆医科大学分子医学与肿瘤研究中心,重庆,400016;第三军医大学基础医学部实验动物学教研室,重庆,400038;河南省新乡医学院第三附属医学院检验科,河南,新乡,543003
基金项目:国家科技攻关计划重点项目 
摘    要:目的 建立稳定表达Bama小型猪细胞色素CYP3A22的HepG2细胞株.方法 通过从Bama小型猪肝组织中提取tRNA、经RT-PCR得到Bama小型猪CYP3A22的基因.并将此基因克隆至亚克隆载体pMD18-T上得到重组质粒pMD-3A22;以测序确定的重组质粒pMD-3A22为模板,采用PCR扩增CYP3A22基因并在其3'添加组氨酸标签,扩增产物经BamH Ⅰ/Xho Ⅰ双酶切,将带有组氨酸标签的CYP3A22基因定向克隆至pcDNA3.1(+)中,测序表明,CYP3A22基因真核表达载体正确构建;将测序正确的CYP3A22基因通过脂质体转染至HepG2细胞,G418筛选10代,RT-PCR及Westernblot分析,并用探针药物硝苯地平对重组细胞进行进行活性鉴定.结果 与HepG2相比,HepG2-CYP3A22细胞株具有极显著的硝苯地平氧化活性.结论 成功建立了稳定表达CYP3A22的HepG2细胞株,可用于相关的药物代谢研究.

关 键 词:Bama小型猪  Sus scrofa CYP3A22  HepG2  RT-PCR  Western blot  探针药物

Establishment of a HepG_2 cell line stably expressing CYP3A22 and identification of the metabolic activity of the probe drugs
XUE Zheng-kai,WEI Hong,SHANG Hai-tao,YANG Gen-qing,YE Bin. Establishment of a HepG_2 cell line stably expressing CYP3A22 and identification of the metabolic activity of the probe drugs[J]. Acta Academiae Medicinae Militaris Tertiae, 2009, 31(16): 1552-1555
Authors:XUE Zheng-kai  WEI Hong  SHANG Hai-tao  YANG Gen-qing  YE Bin
Affiliation:XUE Zheng-kai1,WEI Hong2,SHANG Hai-tao2,YANG Gen-qing3,YE Bin1 (1Research Center for Molecular Medicine and Tumor,Chongqing Medical University,Chongqing 400016,2Laboratory Animal Center,College of Basic Medical Sciences,Third Military Medical University,Chongqing 400038,3Clinical Laboratory,Third Affiliated Hospital of Xingxiang Medical University,Xingxiang 453003,Henan Province,China)
Abstract:Objective To establish a HepG 2 cell line,which can stably express Sus scrofa CYP3A22,for the evaluation of the drug metabolic characteristics of CYP3A22. Methods Sus scrofa CYP3A22 gene obtained by PCR was cloned into the eukaryotic expression vector pcDNA3.1 [The tissue of Bama minipig was amplified by RT-PCR. The gene was subcloned into plasmid pMD18-T (designated as pMD-3A22),and then the gene amplified by recombinant plasmid was designated as pcDNA-3A22]. Then Sus scrofa CYP3A22 gene was confirmed by s...
Keywords:Sus scrofa CYP3A22  HepG2  RT-PCR  Western blot  Bama minipig  Sus serofa CYP3A22  HepG2  RT-PCR  Western blot  probe drug
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