论文部分内容阅读
目的:研究雌激素受体新突变Arg548/Cys548对受体介导的下游基因表达调控的影响,并探讨其机理和在女孩性早熟中的作用。方法:利用已构建的雌激素受体反应元件报道质粒pGL3-promoter-ERE,分别与野生型ESR1表达质粒PSG5-wtER和定点突变受体表达质粒PSG5-mutER共转染CMF-7和MDA-MB-231细胞,转染后加入雌激素、类似物和拮抗物处理,检测转染细胞的荧光素酶活性值。结果:转染突变型受体的CMF-7和MDA-MB-231细胞的荧光素酶活性值明显升高,在生理浓度雌激素及类似物植物类雌激素异黄酮刺激下,Cys548突变型受体基因转染比野生型受体转染的CMF-7和MDA 231细胞均表现出显著强烈的增加萤火虫荧光素酶的活性,表现高敏感的特性;单独加入TAM后荧光素酶活性值没有变化,而同时加入E2和TAM后荧光素酶活性值比加入E2的荧光素酶活性值则明显下降。结论:Cys548突变受体在体外具有较高的敏感性,对药物雌二醇及类似物反应敏感,拮抗剂TAM对受体的活性没有抑制作用,却能竞争性降低雌激素及类似物的作用。
OBJECTIVE: To study the effect of Arg548 / Cys548, a novel estrogen receptor mutation, on the regulation of receptor-mediated downstream gene expression and its mechanism and its role in precocious puberty in girls. METHODS: Plasmid pGL3-promoter-ERE was used to construct a reporter plasmid for estrogen receptor (ERG). CMF-7 and MDA-MB were co-transfected with PSG5-wtER and PSG5- -231 cells. After transfection, estrogen, analogs and antagonists were added to detect the luciferase activity of transfected cells. Results: The luciferase activity of CMF-7 and MDA-MB-231 cells transfected with mutant receptor was significantly increased. Under the stimulation of phytoestrogen isoflavones with physiological concentrations of estrogen and its analogues, Cys548 mutant The gene transfection of CMF-7 and MDA-231 cells transfected with wild-type receptor both showed a significant increase of firefly luciferase activity and showed a high sensitivity; there was no change in luciferase activity after TAM alone , While the addition of E2 and TAM luciferase activity than the value of the addition of E2 luciferase activity was significantly decreased. CONCLUSION: Cys548 mutant receptor has high sensitivity in vitro and is sensitive to the drugs estradiol and its analogues. The antagonist TAM has no inhibitory effect on the activity of the receptor, but it can competitively reduce the effects of estrogen and its analogues .