以PPARγ2为靶点的传统中药细胞学筛选方法的建立和应用

Establishment and application of Chinese herbal cytological screening method targeting at PPARγ2

目的构建含人PPARγ2基因启动子的荧光素酶表达质粒,通过建立稳定转染该质粒的细胞系,建立传统中药细胞学筛选方法。方法首先以p GL3-Basic-Luc和p GL3-Enhancer-Luc为载体构建含不同长度人PPARγ2基因启动子序列的荧光素酶表达质粒。脂质体转染,建立稳定转染上述质粒的3T3-L1细胞系。选取红花和茜草等28种可能具有减肥作用的中药制备成中药水溶性提取物(ECMH),观察不同浓度ECMH对3T3-L1细胞中荧光素酶表达的影响。结果 p GL3-Enhancer-PPARγ2 625 bp-Luc质粒稳定转染的细胞荧光素酶表达最高,是本底的200倍左右。故采用此细胞系进行传统中药筛选,红花ECMH在10、100和1 000μg/m L时均可使3T3-L1细胞中荧光素酶的表达明显增加,分别为对照组的1.63倍、1.90倍和2.30倍(P<0.05)。在10~1 000μg/m L荷叶、茜草ECMH也均能促进3T3-L1细胞中荧光素酶的表达,在浓度为1 000μg/m L时荷叶和茜草对3T3-L1细胞中荧光素酶的表达作用最强,分别为对照组的2.03倍(P<0.01)和2.00倍(P<0.01)。结论成功构建p GL3-Enhancer-PPARγ2625 bp-Luc质粒,并建立稳定转染的3T3-L1细胞系。红花、荷叶和茜草的ECMH能明显促进3T3-L1细胞中人PPARγ2基因启动子的活性,它们可能成为未来潜在的减肥药物。

Objective To establish a natural Chinese herbal cytological screening method through the establishment of stably transfected 3 T3-L1 cell line with the luciferase reporter gene expression plasmid containing human PPARγ2 gene promoter.Methods The luciferase reporter gene expression plasmids containing various lengthPPARγ2 gene promoter sequence were constructed by using p GL3-Basic-Luc and p GL3-Enhancer-Luc as vector.The stably transfected 3 T3-L1 cell line was stablished,and twenty-eight kinds of extracts of Chinese medical herbs（ECMH） were selected.Finally the effects of these ECMH on the luciferase expression in 3 T3-L1 cells were observed.Results The luciferase expression of 3 T3-L1 cells stably transfected with p GL3-Enhancer-PPARγ2 625 bp-Luc plasmid was the highest.Therefore,this cell line was selected for the following screening experiment.Our results showed that the administration of safflower ECMH（10,100,and1 000 μg/m L） significantly promoted the luciferase expression of 3 T3-L1 cells.it was 1.63,1.90,and2.30（P〈0.05） of no drug intervention cells.Similarity,Leaves and madder ECMH（10 - 1 000 μg/m L） also notably increased the luciferase expression of 3 T3-L1 cells.The highest actions were observed,at a concentration of 1 000 μg/m L,which were 2.03（P〈0.01） and 2.00 times of the control group（P〈0.01）,respectively.Conclusions Using this cell line safflower,leaves,madder ECMH were found to significantly promote human PPARγ2 gene promoter activities with the screening method,and they may become potential weight loss medicine in the future.