摘要
目的针对CCAAT增强子结合蛋白α(C/EBPα)启动子,建立能在糖皮质激素下促进间充质干细胞成骨分化的药物筛选体系。方法采用PCR法从小鼠基因组中扩增C/EBPα启动子(-1381 bp/+45 bp)序列,连接pGL3-basic载体构建出pGL3-C/EBPα。利用脂质体将pGL3-C/EBPα与内参载体pRL-SV40共转染293T细胞建立筛药体系。利用地塞米松及氯化锂(LiCl)初步验证了筛药体系的有效性,并测试不同剂量淫羊藿苷、补骨脂素及丹参酮ⅡA在地塞米松作用下对C/EBPα启动子的抑制效应。通过ALP活性及茜素红染色检测这3种药物是否能挽救地塞米松抑制的成骨分化。结果成功构建了基于C/EBPα启动子的药物筛选体系。初步检测发现通过测定荧光素酶活性能反映出地塞米松及LiCl对C/EBPα启动子的调控。淫羊藿苷呈浓度依赖性地抑制地塞米松对C/EBPα启动子的激活(P<0.01或P<0.05),丹参酮ⅡA在中、高剂量时才表现出抑制效应(P<0.05),而补骨脂素未能抑制C/EBPα启动子的激活(P>0.05)。成骨分化实验发现,中、高剂量淫羊藿苷能有效挽救地塞米松抑制的成骨分化(P<0.01),中、高剂量丹参酮ⅡA展示出部分缓解效应(P<0.01),而补骨脂素在地塞米松作用下未能促进成骨细胞分化(P>0.05)。结论经体外实验成功建立了一种激素型骨质疏松症药物筛选体系。
Objective To establish a drug screening system based on CCAAT/enhancer binding protein α(C/EBPα) promoter for screening drugs that can promote mesenchymal stem cells osteogenesis induced by glucocorticoid.Methods DNA sequence of C/EBPα promoter(-1381 bp/+45 bp) was amplified by PCR and subcloned into pGL3-basic vector to establish pGL3-C/EBPα,which was co-transfected with pRL-SV40 into 293 T cells with liposome.The effectiveness of the drug screening system was tested by dexamethasone and LiCl.The inhibitory effects of different doses of icariin,psoralen and tanshinoneⅡA on C/EBPα promoter induced by dexamethasone were measured by this drug screening system.ALP activity assay and Alizarin red staining were used to detect the effect of drugs on dexamethasone-inhibited osteogenesis.Results The drug screening system based on C/EBPα promoter was successfully established.Preliminary detection showed that the regulation on C/EBPα promoter by dexamethasone and LiCl could be sensitively reflected by luciferase activity.Icariin significantly inhibited the active effect of dexamethasone on C/EBPα promoter in a does-dependent manner(P<0.01 or P<0.05).The medium and high doses of tanshinoneⅡA partly inhibited C/EBPα promoter activity(P<0.05),whearas psoralen failed to inhibit the activation of C/EBPα promoter(P>0.05).Osteogenic assay showed that medium and high doses of icariin could effectively rescue the inhibitory effect of dexamethasone on osteogenic differentiation(P<0.01),while which of tanshinoneⅡA represented partial inhibitory effect(P<0.01).However,psoralen failed to promote osteoblast differentiation induced by dexamethasone(P>0.05).Conclusion The effective and sensitive drug screening system for steroid-induced osteoporosis has been successfully established in this study.
出处
《江苏医药》
CAS
2019年第3期217-221,F0002,共6页
Jiangsu Medical Journal
基金
国家自然科学基金(81660376)
贵州省教育厅青年科技人才成长项目[黔教合KY字(2016)194]
遵义医学院大学生创新训练计划项目[遵义科院(2015)3117]
