文献类型：期刊文献

中文题名：以MDR1基因启动子为靶点的药物筛选模型的建立

英文题名：Establishment and Application of a Screening Assay Based on MDR1 Prompter

作者：李长龙[1,2];萨晓婴[2];邓皖利[3];许建华[3];范忠泽[3];刘建文[1]

机构：[1]华东理工大学药学院,上海200237;[2]浙江省医学科学院,浙江省实验动物中心,杭州310013;[3]上海中医药大学附属普陀中心医院,上海200062

年份：2008

卷号：34

期号：5

起止页码：650

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2004】；CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：MDR1基因;双荧光素酶报告基因;药物筛选

外文关键词：MDR1 gene; dual-luciferase report gene;drug screening

摘要：通过构建以MDR1启动子为启动序列的荧光素酶报告基因载体,建立基于双荧光素酶报告基因系统的多药耐药抑制剂筛选模型。从HCT-8细胞中提取DNA并克隆含有MDR1基因启动子的序列。将该序列重组到荧光素酶报告基因载体pGL-3-Basic的启动区域中,从而构建报告基因载体pGL-MDR1。将pGL-MDR1和pRL-TK载体共转染到HCT-8和HCT-8/VCR细胞中,建立用于筛选多药耐药抑制剂的方法。通过调节不同载体的比例来优化转染效率。通过MDR1基因激活剂(热诱导)和抑制剂(EGCG)来验证该方法。通过直接测序法验证了pGL-MDR1含有MDR1基因启动子序列且没有出现碱基突变。在n(pGL-MDR1)∶n(pRL-TK)=5∶5时,转染效率最高并具有最高的荧光素酶活性。通过MDR1基因激活处理后表现为时间依赖性地激活MDR1基因的表达,而MDR1基因抑制剂的作用则相反。
To construct the recombinant luciferase report gene vector contained MDR1 gene promoter and establishment screening assay based on MDR1 prompter, total DNA was extracted from HCT-8 cells and it was used for amplifying the fragment contained MDR1 gene promoter by PCR. The amplified product was inserted into pGL-3-Basic vector. The PCR product and pGL-MDR1 vector were verified by sequencing and alignment. The pGL-MDR1 and pRL-TK vector were co-transfected into HCT-8 and HCT- 8/VCR cells. The activity of MDR1 gene promoter could be assayed by measured luciferase. The method was optimized by changing ratio of two vectors. The results showed that the highest transfection efficiency and luciferase activity were found in ratio of n（pGL-MDR1） ： n（pRL-TK）=5 ： 5. Moreover, the inductor （hyperthermia） and inhibitor （EGCG） of MDR1 gene were used for authenticating the method. The results showed that hyperthermia and EGCG significantly induced or inhibited activity of MDR1 prometor in HCT-8 or HCT-8/VCR cells, respectively.