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目的:改良E-screen实验,提高其筛选具有雌激素样作用物质的准确性.方法:构建雌激素受体反义RNA表达质粒pCASER,以脂质体转染MCF-7细胞,G418筛选阳性克隆.PCR检测雌激素受体的DNA是否整和地MCF-7细胞的基因组DNA中,Westernblot检测雌激素受体的表达;MTT法检测细胞的增殖.结果:筛选出一株雌激素受体反义RNA表达克隆(MTASER).17β-雌二醇,金雀异黄素,Dro-loxifen,Miyabenol和Kobophenol在一定浓度均可促进MCF-7细胞增殖,且对MCF-7的促增殖作用大于对MTASER的促增殖作用;表皮生长因子对两株细胞的促增殖作用的差别无显著意义.结论:改良的E-screen实验筛选具有雌激素样作用物质的准确性高于通常的E-screen实验.
Objective: To improve the accuracy of screening estrogen-like substances by E-screen experiment.Methods: The estrogen receptor antisense RNA plasmid pCASER was constructed and transfected into MCF-7 cells by lipofectamine 2000. The positive clones were screened by G418 The expression of estrogen receptor in MCF-7 cells was detected by PCR, and the proliferation of MCF-7 cells was detected by MTT assay.Results: The antisense of estrogen receptor 17β-estradiol, genistein, Dro-loxifen, Miyabenol and Kobophenol could promote the proliferation of MCF-7 cells at a certain concentration, and the effect on the proliferation of MCF-7 was greater than that of MTASER Of epidermal growth factor on the proliferation of two cells was no significant difference.Conclusion: The modified E-screen screening screening estrogen-like substances with higher accuracy than the usual E-screen experiments.