PPARδ激动剂高通量筛选模型的建立

Establishment of a Cell-based High-throughput Screening Model for PPARδ Agoni sts

为建立基于细胞瞬时转染的过氧化物酶体增殖物激活受体δ(peroxisome proliferator-activated receptor delta PPARδ)激动剂高通量筛选模型,用RT-PCR技术从肝的总RNA中扩增PPARδ基因序列,将其连至T克隆载体进行测序。将序列正确的PPARδ片段连接至pTARGET载体上构建表达载体pTARGET-ppARδ;将合成的3个拷贝的PPRE(peroxisome proliferator receptorresponse element)插入pGL3-promoter构成报告质粒pGl3-PPRE×3-luc。用脂质体转染技术将表达载体与报告质粒共转染细胞系,通过检测荧光素酶基因的表达状况评价化合物对PPARδ的激动活性。通过多种条件的优化,得到了最佳的共转染条件。阳性药苯扎贝特明显提高荧光素酶的表达,最大上调倍增数可达10倍,并且在一定浓度下阳性药与相对荧光酶的活性表达有较好的量效关系。该筛选模型灵敏、稳定,为对PPARδ激动剂进行药物研发和PPARδ机理的研究打下基础。

To establish a new high-throughput screening model for the agonist of PPARδ, PPARδ gene was obtained by reverse transcriptase-polymerase chain reaction （RT-PCR）, and subcloned to pGEM-T Vector for sequencing, then the PPARδ fragment was excised by restriction enzymes, and inserted into pTARGETTM Vector to construct expression vector pTARGETTM-ppARδ. Insert three copies of PPRE into pG13-promoter vector to construct expression vector pG13-PPRE ×3-luc. The vector pTARGET^TM-ppARδ was transiently cotransfected with pG13-PPRE × 3-1uc into different cell lines to assay the expression levels of luciferase. The PPARδ agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARδ, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.