基于COL1A1启动子的抗肝纤维化药物高通量筛选模型的建立及应用

发布时间：2018-05-05 04:25

本文选题：肝纤维化 + 荧光素酶报告基因　；参考：《药学学报》2015年02期

【摘要】：为了有效筛选潜在的抗肝纤维化药物,本实验室建立了基于COL1A1启动子的高通量筛选细胞模型。该细胞模型在TGF-β1的刺激下,激活COL1A1启动子及荧光素酶报告基因的表达,而该激活作用可以被候选化合物抑制。作者首先构建COL1A1启动子和荧光素酶报告载体p GL4.17的重组质粒。采用双荧光素酶报告基因检测系统对该启动子的活性进行检测,发现重组质粒比对照组荧光素酶活性高15.98倍。该质粒转染到人肝星状细胞株LX2中,用无限稀释法得到单克隆细胞株,并利用活体成像仪筛选出稳定表达COL1A1启动子的单克隆细胞株LX2-COL。经TGF-β1诱导后检测该单克隆细胞株LX2-COL对候选化合物(S1~S7)的反应性,其中S7对启动子活性抑制作用最强。Real-time RT-PCR和Western blotting验证了经该细胞模型筛选得到的化合物S7能够抑制Ⅰ型胶原m RNA和蛋白水平的表达。结果表明,该细胞模型的建立能够实现对潜在的抗肝纤维化化合物的高通量筛选。
[Abstract]:In order to screen potential antifibrotic drugs effectively, a high throughput screening cell model based on COL1A1 promoter was established. The cell model stimulated by TGF- 尾 1 activates the expression of COL1A1 promoter and luciferase reporter gene, which can be inhibited by candidate compounds. The authors first constructed the recombinant plasmid of COL1A1 promoter and luciferase report vector p GL4.17. Double luciferase reporter gene detection system was used to detect the activity of the promoter. It was found that the activity of the recombinant plasmid was 15.98 times higher than that of the control group. The plasmid was transfected into human hepatic stellate cell line LX2. The monoclonal cell line LX2-COL, which expressed COL1A1 promoter stably, was obtained by infinite dilution method. After induction by TGF- 尾 1, the reactivity of the monoclonal cell line LX2-COL to the candidate compound S1, S7, was detected. Among them, S7 had the strongest inhibitory effect on promoter activity. Real-time RT-PCR and Western blotting confirmed that S7 could inhibit the expression of type I collagen m RNA and protein. The results showed that the cell model could achieve high throughput screening of potential anti-hepatic fibrosis compounds.
【作者单位】：中国医学科学院、北京协和医学院医药生物技术研究所;
【基金】：国家自然科学基金资助项目(81170409) 中国肝炎防治基金会-王宝恩肝纤维化研究基金资助课题(20110026) 国家“重大新药创制”科技重大专项(2012ZX09301002-001)
【分类号】：R963

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