以Apo A-I为靶点的基因表达上调剂筛选模型的建立

Establishment of a high-throughput screening model for identifying up-regulator of human Apo A-I expression

目的建立靶向人ApoA-I转录调控序列的高通量药物筛选模型,用于筛选ApoA-I基因表达上调剂。方法以人基因组DNA为模板,PCR扩增ApoA-I上游的启动子序列,克隆至荧光素酶报告基因质粒pGL4.17上游,采用脂质体介导的方法将重组质粒转染人肝癌细胞HepG2,在G418抗性存在的条件下,通过有限稀释法筛选稳定转染细胞株。对溶剂浓度和药物作用时间进行条件优化,应用阳性化合物染料木素评价模型有效性,通过检测荧光素酶的表达活性变化来筛选人ApoA-I基因表达上调剂。结果通过PCR成功扩增了人ApoA-I基因上游的启动子序列,构建了相应的重组荧光素酶报告基因质粒pGL4-ApoP,并建立稳定转染细胞株ApoP-LucHepG2。对筛选条件优化后,应用阳性化合物进行评价,筛选窗相关系数Z’因子为0.68,大于0.5,适于进行高通量筛选。应用该筛选模型对5000余种化合物进行筛选,4个黄酮类化合物和白藜芦醇显示出较好的活性,半数有效浓度均小于10μg/ml。结论成功建立了人ApoA-I基因表达上调剂筛选模型,并利用该模型筛选到5个活性化合物。

Objective To establish a high-throughput screening model based on transcriptional regulation of human apolipoprotein A-I for identifying up-regulator of Apo A-I gene expression.Methods Human genomic DNA was used as a template to amplify the promoter sequence of Apo A-I using PCR.The fragment was cloned into pGEM-T vector and characterized by sequencing.The promoter fragment was then inserted upstream of luciferase reporter gene of pGL4.17,and then transfected into HepG2 cell line using LipofectamineTM 2000.Stable transfectant was obtained using limited cell dilution method in the presence of G418.Samples were detected by measuring luciferase activity of stable transfected HepG2 cells in 96-well format after assay optimization and validation with genistein.Results The promoter sequence of Apo A-I containing essential transcriptional elements was obtained from human genomic DNA by PCR.The reporter plasmid named pGL4-ApoP was constructed by inserting the promoter sequence upstream of luciferase reporter gene of pGL4.17.The drug screening model was established and evaluated using genistein as a positive control.Z’-factor is above 0.5,showing that this model was robust and reliable.About 5000 compounds were screened and resveratrol and several flavonoids were identified as positive compounds.Conclusion This new drug screening model could be efficiently applied to screen up-regulators of human Apo A-I gene expression.