调节维甲酸受体稳定性的化合物细胞筛选体系的建立

Establishment of cell-based screening system for compound regulating the stability of retinoic acid receptors

目的·构建可用于发现调节维甲酸受体(retinoic acid receptors,RARα)蛋白稳定性的化合物的细胞筛选体系。方法·对pMSCV质粒进行改造,插入绿色荧光蛋白(EGFP)-RARα融合基因和红色荧光蛋白(Ds Red)基因,两者之间以内部核糖体结合位点(IRES)序列分隔;构建成功的质粒稳定转染急性早幼粒细胞白血病细胞NB4,流式细胞术分析全反式维甲酸、丙戊酸钠、阿糖胞苷、来那度胺、依托泊苷、孟鲁司特纳及藤黄酸处理细胞后EGFP与DsRed的荧光信号的变化,间接反映RARα蛋白表达水平;并通过Western blotting实验进一步验证阳性化合物对RARα蛋白水平的影响。结果·成功构建了蛋白稳定性双荧光筛选体系,并发现丙戊酸钠可有效使RARα蛋白的表达水平稳定。结论·双荧光筛选系统可用于调节RARα蛋白稳定性的化合物的筛选。丙戊酸钠是一个新的稳定RARα的化合物。该方法对建立稳定其他蛋白的化合物筛选系统具有参考价值。

Objective · To establish a cell-based screening system for identification of compounds with activity in regulating retinoic acid receptor （RARu） stability. Methods · The modified pMSCV plasmid constructs, named as RARu-EGFP-IRES-DsRed, consists of enhanced green fluorescent protein （EGFP） fusing to RARa and red fluorescent protein （DsRed） as internal references incorporating the internal ribosome entry site （IRES） as interval sequence. The RARct-EGFP-IRES-DsRed plasmid was stably transfected into NB4 cells which were named as NB4-pMGIR-RARu. Fluorescence signals of EGFP and DsRed indirectly reflecting the expression of RARα, were detected by flow cytometry in cells that were treated with all-trans retinoic acid, sodium valproate, cytarabine, lenalidomide, etoposide, montelukast and gambogic acid, respectively. Effects of these compounds on the expression of RARu protein were further examined by Western blotting. Results · A double fluorescence reporter system for screening compounds that can increase the stability of RARer protein was successfully established, and sodium valproate was identified as a potent compound to promote the stability of RARα. Conclusion· The double fluorescence reporter system can be used to screen compounds regulating the stability of RARu protein, which can be further used to identify compounds regulating the stability of other proteins.