Establishment And Evaluation Of The High Throughput Screening System For Antagonistic Agents Of Scar Hypertrophy Based On Smad3

ObjectiveThe study is to construct a high throughput screening system for antagonistic agents of scar hypertrophy based on smad3.MethodsA weak promoter SV40 was obtained by PCR from plasmid pGL2-control. After purified the promoter was used to replace the CMVIE promoter in the pEGFP-N1 plasmid and gained pSV40-EGFP plasmid. Two shares of pSV40-EGFP were ready, one share was digested by XhoⅠand HindⅢand a backbone with EGFP was obtained. The other was digested by BglⅡand HindⅢin order to get a fragment with SV40 promoter. The two fragments and a oligodeoxynucleotide of COL1A2 motif were linked together and generated p4COL1A2-EGFP plasmid which contained the backbone with EGFP, the fragment with SV40 promoter and artificial synthetic 4 copies of COL1A2 motif which acted as the cis-acting element and possessed XhoⅠand BglⅡsites. After the EGFP fragment of the p4COL1A2-EGFP plasmid was substituted with d2EGFP, reporter plasmid p4COL1A2-d2EGFP was finally constructed. The plasmid p4COL1A2-d2EGFP was transfected into the mouse embryo fibroblasts (NIH3T3 cells). After selected with G418, reporter cell line which was based on smad3 was gained and named NIH3T3-d2EGFP. TGF-β1 was added to NIH3T3-d2EGFP cell line which was cultured in DMEM culture medium. The expression of d2EGFP among different time points was detected through fluorescence microscope and flow cytometry.The smad3 transcription factor decoy (TFD) oligodeoxynucleotides were synthesized in form of single strand. After renaturation, a nicked dumbbell DNA oligonucleotide containing a smad3 binding site was obtained. TGF-β1 was added to NIH3T3-d2EGFP which had been transfected with smad3 transcription factor decoy (TFD) oligonucleotides...