Establishment and application of a high-throughput drug screening model based on COL1A1 promoter for anti-liver fibrosis.
- Author:
Shuang-Shuang ZHAO
;
Ju-Xian WANG
;
Yu-Cheng WANG
;
Rong-Guang SHAO
;
Hong-Wei HE
- Publication Type:Journal Article
- MeSH:
Collagen Type I;
genetics;
Drug Evaluation, Preclinical;
methods;
Genes, Reporter;
Hepatic Stellate Cells;
High-Throughput Screening Assays;
Humans;
Liver Cirrhosis;
drug therapy;
Luciferases;
Plasmids;
Promoter Regions, Genetic;
RNA, Messenger;
Transfection;
Transforming Growth Factor beta1;
pharmacology
- From:
Acta Pharmaceutica Sinica
2015;50(2):169-173
- CountryChina
- Language:Chinese
-
Abstract:
For screening the potential drugs as anti-liver fibrosis candidates, we established a high- throughput drug screening cell model based on COL1A1 promoter. The activity of COL1A1 promoter and luciferase reporter gene can be elevated by TGF-β1, and inhibited by candidate drugs. We constructed a recombined plasmid with COL1A1 promoter and luciferase reporter gene pGL4.17, the activity of COL1A1 promoter was reflected by fluorescence intensity. COL1A1 promoter activity was detected by Dual-Luciferase Reporter Assay System, it came that the relative luciferase activity of COL1A1 promoter was 15.98 times higher than that of control group induced by TGF-β1, showing the recombined plasmid could be used in cell model. The recombined plasmid was transfected into human hepatic stellate cells LX2, detected the effect of potential drugs, and obtained a stable expression system through stable transfection and monoclonal cell culture. A sample which could reduce COL1A1 promoter activity signally by our cell model, decreased collagen I mRNA and protein expression detected by real-time RT-PCR and Western blotting. It indicates this novel cell model can be used in high-throughput drug screening of potential anti-liver fibrosis drugs.
