葛根素对子宫内膜RL95-2细胞P450芳香化酶表达的调控作用.docVIP

　　葛根素对子宫内膜RL95?2细胞P450芳香化酶表达的调控作用 【摘要】 探讨葛根素对子宫内膜RL95?2细胞P450芳香化酶（aromatase P450, P450arom）表达的影响及其对RL95?2细胞P450arom基因表达的调控作用。 方法：将梯度稀释的葛根素在不同时间点加入RL95?2细胞中，分别采用实时聚合酶链反应（real?time polymerase chain reaction, RT?PCR）检测药物对RL95?2细胞P450arom mRNA表达水平的影响。以人类基因组DNA为模板扩增人P450arom组织特异性启动子2（PⅡ）转录起始位点上游序列。PCR产物定向克隆到报告基因载体pGL3?Basic中，构建人P450arom PⅡ系列报告基因质粒，并经限制性内切酶消化鉴定和基因测序证实。瞬时转染系列重组质粒至RL95?2细胞，加入葛根素刺激，分析相关转录因子，利用RT?PCR及aterial and methods 2.1 Materials and cell culture Puerarin Sigma (St. Louis, MO). Luciferase Assay Systems and pGL3?Basic Vector Promega (Madison, D). All chemicals and enzymes Sigma (St. Louis, MO). Phenol red?free (PRF) DMEM/F12, and charcoal?stripped fetal bovine serum (CS?FBS) Gibco (Grand Island, NY). RL95?2 (ATCC CRL?1671TM, USA) human endometrium carcinoma cells ented idified atmosphere of 95% air and 5 %CO2, at 37 ℃. 2.2 RNA isolation and quantitative RT?PCR analysis RL95?2 cells ately 70% to 80% confluence prior to the mencement of each experiment. Total RNA 2 g of total RNA primers folloanufacturers protocol (MBI Fermantas, Vilnius, Lithuania). The single?stranded cDNA e PCR to evaluate the relative expression levels of P450arom (5?GAGAGCATACAGAAGATACACGACT?TG and 5?GACCAGCCTTCTCTAGTGTTCCA) and human c?jun (5?CCCCCAGCGTATCTATATGGAA and 5? GCTGTCCCTCTCCACTGCAA) pared an β?actin (5?CTGGAACGGTGAAGGT?GACA and 5?AAGGGACTTCCTGTAACAATGCA). RT?PCR ed according to the ABI manufacturers protocols (Applied Biosystems, CA). All samples ined in triplicate. PCR?specific amplification ed in the Applied Biosystems (ABI 7300) quantitative RT?PCR machine. The reactions ethod［17］. 2.3 SF); 1 mmol/L ethylenediamine tetraacetic acid (EDTA)］. This in incubation on ice and centrifugation at 10 000×g for 10 min at 4 ℃. Protein concentr?ations ined by a modified Bradford assay (Bio?Rad Laboratories, Inc., Hercules, CA). Protein extracts ide gel, blotted onto a nitrocellulose membrane (Millipore Corp, Bedford, USA), and incubated (Biovision, USA) and anti?β?actin (Cel