基于Smad3的抗瘢痕增生药物高通量筛选体系的构建及效果评价

Establishment and effect evaluation of the high throughput screening system for antagonistic agents of scar hypertrophy based on smad3

目的:构建Smad3反应性不稳定增强型绿色荧光蛋白(d2EGFP)报告系统,作为抗瘢痕增生药物高通量筛选体系。方法:分别以EGFP、d2EGFP为报告基因,neor为筛选基因,构建以4×成纤维细胞α2Ⅰ型前胶原(COL1A2)基序为增强子、SV40为启动子的报告基因载体p4COL1A2-EGFP和p4COL1A2-d2EGFP,并分别稳定转染NIH3T3细胞,获得Smad3反应性报告细胞株NIH3T3-d2EGFP和NIH3T3-EGFP。分别向两种细胞株中加入TGF-β1,比较不同时相点d2EGFP和EGFP的表达。应用Smad3“圈套”寡核苷酸(TFD)转染NIH3T3-d2EGFP,加入TGF-β1,观察TFD对TGF-β1诱导d2EGFP表达的拮抗作用。结果:NIH3T3-d2EGFP是较理想的Smad3反应性报告细胞株,Smad3TFD对TGF-β1诱导调控d2EGFP的表达具有明显的拮抗作用。结论:Smad3反应性d2EGFP报告系统可特异、灵敏、动态地反映和监测Smad3的活性变化。

Objective To construct smad3-responsive destabilized enhanced green fluorescent protein （d2EGFP） reporter system, which was employed as the high throughput screening system of antagonistic agents of scar hypertrophy. Methods Two vectors, p4COL1A2-d2EGFP containing d2EGFP reporter gene and p4COL1A2-EGFP with EGFP gene, were constructed with 4 copies of COL1A2 motif as enhancer, SV40 as basic promoter and neor gene as selective gene. The smad3-responsive clonal cell lines named NIH3T3-d2EGFP and NIH3T3-EGFP were established after the two vectors stably being transfected into NIH3T3 cells respectively. The expression of the EGFP and d2EGFP was compared among different time points after the stimulation of TGF-β1. The change of fluorescence intensity was observed after TGF-β1 was added to NIH3T3-d2EGFP which had been transfected with smad3 transcription factor decoy （TFD） oligonucleotides for half an hour, Results p4COL1A2-d2EGFP was the better smad3-responsive d2EGFP reporter gene system and the smad3 TFD could antagonize the d2EGFP expression induced by TGF-β1 significantly. Conclusion The smad3-responsive d2EGFP reporter system could report and monitor the change of smad3 activity specifically, sensitively and dynamically.