PPARγ激动剂高通量筛选模型的构建和应用

发布时间：2018-12-14 01:08

【摘要】：过氧化物酶体增殖物激活受体y (PPARy)属于核受体超家族成员,是一种配体激活型转录因子,在糖脂代谢、炎症和癌症中起着关键作用。在人体脂肪组织细胞,巨噬细胞及肝、肺等脂肪贮存细胞中均有不同程度的PPARy表达活性。当有配体存在时,被激活的PPARy可以发挥促进脂肪细胞的生成和分化,缓解胰岛素抵抗,负向调控炎症反应,调节肿瘤的发生发展及延缓组织器官纤维化等作用,与多种疾病的发生发展都有着密切的关系。随着高通量药物筛选技术的不断发展,基于靶点或者靶信号的药物筛选逐步成为药物研发的最有效方式之一。因此,构建一种基于PPARy信号通路的高通量药物筛选模型,并应用于PPARy激动剂的筛选,将会为心血管相关疾病的治疗提供更多的候选药物,开拓疾病治疗的新途径。本课题构建了一种基于PPARy信号通路的高通量药物筛选模型,并将其应用于天然单体化合物的筛选,同时对得到的先导化合物做了初步探究。我们通过择优选择PPARy组成型活化细胞,并对PPARy响应元件PPRE比例进行多次调整,最终得到药物筛选模型细胞CLPPD。该模型以Chang Liver为宿主细胞,以荧光素酶为报告基因,含有23个PPRE重复序列的响应元件及完整PPARy表达元件。通过DNA测序、Western-Blot实验及传代实验证实该细胞模型成功插入目的元件并稳定性良好,能够应用于PPARy激动剂的筛选。随后我们筛选了2500余种天然单体化合物,复筛得到了5种有信号激活作用的先导化合物,其中以15#化合物作用最佳。通过MTT方法及荧光活性检测,我们发现15#化合物细胞毒性小,且相比于对照在15μM处理浓度下有2倍的PPARy激活作用。Western-Blot实验及半定量RT-PCR实验进一步证实,15#化合物可提高PPARy在CLPPD细胞和THP-1细胞中的表达活性。综上所述,我们成功构建了一种PPARy激动剂的高通量筛选模型,并将其应用于2500余种天然产物的筛选,最终得到5种先导化合物,进一步研究发现15#化合物能显著激活PPARy,并促进PPARy的表达,为进一步筛选得到PPARy激动剂,寻找心血管疾病靶向药物提供了一定的技术和理论依据。
[Abstract]:Peroxisome proliferator activated receptor (y (PPARy), a member of nuclear receptor superfamily, is a ligand activated transcription factor, which plays a key role in glycolipid metabolism, inflammation and cancer. In human adipose tissue cells, macrophages, liver, lung and other fat storage cells have different degrees of PPARy expression activity. In the presence of ligands, activated PPARy can promote adipocyte formation and differentiation, alleviate insulin resistance, negatively regulate inflammatory response, regulate tumorigenesis and progression, and delay fibrosis of tissues and organs. It is closely related to the occurrence and development of many diseases. With the development of high throughput drug screening technology, drug screening based on target or target signal has gradually become one of the most effective methods of drug development. Therefore, the establishment of a high-throughput drug screening model based on PPARy signaling pathway and its application to the screening of PPARy agonists will provide more candidate drugs for the treatment of cardiovascular diseases and open up new approaches to the treatment of cardiovascular diseases. In this paper, a high throughput drug screening model based on PPARy signaling pathway was constructed and applied to the screening of natural monomers. We selected the PPARy constitutive activated cells selectively, and adjusted the PPRE ratio of PPARy response elements many times. Finally, we obtained the drug screening model cells CLPPD.. The model uses Chang Liver as host cell and luciferase as reporter gene. It contains 23 response elements of PPRE repeat sequence and complete PPARy expression element. The DNA sequencing, Western-Blot and passage experiments confirmed that the cell model was successfully inserted into the target element and stable, and could be applied to the screening of PPARy agonists. Then we screened more than 2500 natural monomers and obtained five leading compounds with signal-activated activity, of which 15# was the best. By MTT method and fluorescence assay, we found that the cytotoxicity of 15# compound was less than that of the control, and the activity of PPARy was twice as much as that of the control at the concentration of 15 渭 M. Western-Blot and semi-quantitative RT-PCR experiments further confirmed the cytotoxicity of 15# compound. Compound 15# could increase the activity of PPARy expression in CLPPD cells and THP-1 cells. To sum up, we successfully constructed a high-throughput screening model for PPARy agonists, and applied it to the screening of more than 2500 natural products. Finally, five leading compounds were obtained. Further studies showed that 15# compounds could significantly activate PPARy,. It can also promote the expression of PPARy and provide some technical and theoretical basis for the further screening of PPARy agonists and the search for targeted drugs for cardiovascular diseases.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R96

【相似文献】