调节维甲酸受体稳定性的化合物细胞筛选体系的建立

发布时间：2018-09-01 10:07

【摘要】：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。
[Abstract]:Objective to construct a cell screening system that can be used to identify compounds that regulate the stability of retinoic acid receptor (retinoic acid receptors,RAR 伪. Methods pMSCV plasmid was modified and inserted into (EGFP) RAR 伪 fusion gene and red fluorescent protein (Ds Red) gene, separated by internal ribosomal binding site (IRES) sequence. NB4, flow cytometry analysis of all trans retinoic acid, sodium valproate, cytarabine, linalamine, etoposide was successfully constructed and transfected into acute promyelocytic leukemia cells by flow cytometry. The changes of fluorescence signals of EGFP and DsRed after treated with mongolu and luteinic acid indirectly reflected the expression level of RAR 伪 protein, and the effect of positive compounds on RAR 伪 protein level was further verified by Western blotting experiment. Results the double fluorescence screening system of protein stability was successfully constructed, and it was found that sodium valproate could effectively stabilize the expression of RAR 伪 protein. Conclusion double fluorescence screening system can be used to screen compounds that regulate the stability of RAR 伪 protein. Sodium valproate is a new stable compound of RAR 伪. This method has reference value for the establishment of screening system for stabilizing other proteins.
【作者单位】： 上海交通大学医学院病理生理学教研室细胞分化与凋亡教育部重点实验室;上海市瑞金康复医院;
【基金】：国家自然科学基金(81570118) 上海市科委资助项目(15401901800) 国家大学生创新性实验计划项目(201110248075)~~
【分类号】：R3411
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本文编号：2216870