人ApoA-I基因表达上调剂筛选模型的建立.pdfVIP

人Apo A-I 基因表达上调剂筛选模型的建立 杜郁，杨媛，司书毅，洪斌 （中国医学科学院北京协和医学院医药生物技术研究所，北京 100050 ） 摘要：目的 建立靶向人载脂蛋白A-I （Apo A-I ）调控序列的药物筛选模型，为筛选新型抗 动脉粥样硬化药物奠定基础。方法 PCR 扩增Apo A-I 上游调控序列，构建重组的荧光素酶 报告基因质粒pGL4-ApoP1～P5 ，选择pGL4-ApoP2 和pGL4-ApoP4 转染人肝癌细胞HepG2 建立稳定转染细胞株，通过检测荧光素酶的表达活性变化来筛选人 Apo A-I 基因表达上调 剂。 结果 利用两个稳定转染细胞株成功建立了人Apo A-I 基因表达上调剂筛选模型，应用 阳性化合物进行评价，Z 因子分别为0.56 和0.68 ，均大于0.5 。 结论 该筛选模型可有效应 用于人Apo A-I 基因表达上调剂的高通量筛选。 关键词：载脂蛋白A-I ；Apo A-I 调控序列；荧光素酶报告基因；药物筛选 Establishment of a Drug Screening Model for Identifying Up-regulator of Human Apo A-I Expression DU Yu, YANG Yuan, Si Shu-yi, HONG Bin (Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China) ABSTRACT: OBJECTIVE To establish a new drug screening model based on transcriptional regulation of human apolipoprotein A-I (Apo A-I) for identifying up-regulator of Apo A-I gene expression. METHODS Five upstream regulatory sequences of Apo A-I were obtained from human genomic DNA by PCR. A serial of reporter plasmids named pGL4-ApoP1~P5 were constructed by inserting the regulatory sequence upstream of luciferase reporter gene of pGL4.17. Two stable transfected HepG2 cell lines harboring pGL4-ApoP2 and pGL4-ApoP4 respectively, were selected using limited cell dilution method in the presence of G418. Samples were detected by measuring luciferase activity of two stable transfected HepG2 cells in microplate format. RESULTS The drug screening models were established and evaluated, Z-factor is above 0.5 using genistein as a positive control. CONCLUSION This new drug screening model is stable and specific, may be efficiently used to screen up-regulators of human Apo A-I gene expression. KEY WORDS: apolipoprotein A-I; Apo A-I regulatory sequence; luciferase reporter gene; drug screening 动脉粥样硬化性（atheroscle