中文摘要：

目的研究雷公藤甲素（triptolide,TP）对人乳腺癌MCF-7细胞系增殖的影响及其与P53、P73基因表达和甲基化状态的关系。方法用不同浓度（10、20、40 ng/ml）TP处理MCF-7细胞,采用MTT法检测TP对MCF-7细胞增殖的作用;RT-PCR检测MCF-7细胞甲基转移酶1（DNMT1）、DNMT3a、DNMT3b mRNA的表达;甲基特异性PCR检测TP对MCF-7细胞P53、P73基因甲基化的影响;蛋白质印迹分析检测MCF-7细胞中P53、P73蛋白的表达。结果 TP剂量依赖性地抑制MCF-7细胞的增殖（P〈0.05,P〈0.01）,其半数抑制浓度（IC50）约为20 ng/ml,高浓度（40 ng/ml）时抑制率达（70.1±3.52）%。TP可明显抑制MCF-7细胞中DNMT1、DNMT3a、DNMT3b mRNA的表达（P〈0.05,P〈0.01）。TP处理MCF-7细胞后P53启动子区的甲基化降低,TP（20 ng/ml）处理后P53 mRNA的表达升高,而在高浓度（40 ng/ml）TP作用下表达明显上调（P〈0.01）;TP处理MCF-7细胞后P73基因启动子区的甲基化则明显降低,且TP（10 ng/ml）处理后P73 mRNA的表达亦明显增强（P〈0.05）,并呈剂量依赖性。蛋白质印迹分析检测TP（20 ng/ml）处理MCF-7细胞后,P53、P73蛋白的表达均增强。结论TP可通过抑制甲基转移酶活性和抑制P53特别是P73基因启动子区甲基化促进P53、P73的表达,并且能够抑制MCF-7细胞的增殖。

英文摘要：

Objective To study the effect of triptolide（TP） on the proliferation of breast carcinoma cell line MCF-7 and its association with P53/P73 gene expression and methylation.Methods MCF-7 cells were treated with different concentrations of TP（10 ng/ml,20 ng/ml,and 40 ng/ml）,and the proliferation of MCF-7 cells was measured by MTT method.The expressions of methyltransferase DNMT1,DNMT3a and DNMT3b mRNA were measured by RT-PCR in MCF-7 cells,P53/P73 gene methylation was analyzed by methylation specific PCR,and the protein expression of p53/P73 in MCF-7 cells was examined by Western blotting assay.Results TP inhibited the proliferation of MCF-7 cells in a dose-dependent manner（P〈0.05,P〈0.01）,with the inhibitory rate being（70.1±3.52）% at 40 ng/ml TP,and the IC50 of TP was 20 ng/ml.TP significantly inhibited DNMT1,DNMT3a,and DNMT3b mRNA expression in MCF-7 cells（P〈0.05,P〈0.01）,and it also significantly inhibited methylation of P53 promoter region.TP increased P53 gene expression at 20 ng/ml and the increase was significant at 40 ng/ml（P〈0.01）.TP reversed the hypermethylation of P73 gene in MCF-7 cells;it also significantly increased P73 mRNA expression at 10 ng/ml（P〈0.05）,and the increase was in a dose-dependent manner.Western blotting analysis showed that TP（20 ng/ml） increased the protein expression of P53 and P73 in MCF-7 cells.Conclusion TP can promote the expression of P53 and P73 genes through inhibiting methyltransferase-dependent gene methylation,and further inhibit the proliferation of MCF-7 cells.