CAJ | 학술논문

目的探讨17β-雌二醇(17β-estradiol,E2)对大鼠泌乳素腺瘤细胞株GH3细胞Calbindin-D9k(CaBP-9k)表达的影响,并探讨选择性雌激素受体拮抗剂ⅡCI 182780是否能够拮抗雌激素对CaBP-9k表达的诱导作用.方法以GH3细胞作为垂体泌乳素腺瘤的体外模型,通过免疫荧光法观察CaBP-9k在GH3细胞的表达情况;E2分3个剂量:10-8、10-9、10-10 M对GH3细胞作用24h,分别以RT-PCR法及Western blot法检测CaBP-9k基因和蛋白的表达水平;E2以10-8 M的剂量对GH3细胞作用24 h,同时设ⅡCI 182780拮抗组(10-6M),被用于研究ER介导的通路对CaBP-9k表达的影响;用免疫共沉淀法检测CaBP-9k与ERα的相互作用关系.结果在GH3细胞中,CaBP-9k的表达水平随E2给药浓度的增高而增加.当同时给予ⅡCI 182780时,E2诱导CaBP-9k表达的作用被拮抗,其表达水平显著降低.进一步通过免疫共沉淀法证实,在GH3细胞中,CaBP-9k与ERα能够直接结合,E2能够上调2者的相互作用.结论雌激素可能通过ERα途径诱导GH3细胞CaBP-9k的表达,且CaBP-9k与ERα能够直接结合相互作用.提示CaBP-9k可能参与GH3细胞ER途径相关的生物学效应.
목적 탐토17β-자이순(17β-estradiol,E2)대대서비유소선류세포주GH3세포Calbindin-D9k(CaBP-9k)표체적영향,병탐토선택성자격소수체길항제ⅡCI 182780시부능구길항자격소대CaBP-9k표체적유도작용.방법 이GH3세포작위수체비유소선류적체외모형,통과면역형광법관찰CaBP-9k재GH3세포적표체정황;E2분3개제량:10-8、10-9、10-10 M대GH3세포작용24h,분별이RT-PCR법급Western blot법검측CaBP-9k기인화단백적표체수평;E2이10-8 M적제량대GH3세포작용24 h,동시설ⅡCI 182780길항조(10-6M),피용우연구ER개도적통로대CaBP-9k표체적영향;용면역공침정법검측CaBP-9k여ERα적상호작용관계.결과 재GH3세포중,CaBP-9k적표체수평수E2급약농도적증고이증가.당동시급여ⅡCI 182780시,E2유도CaBP-9k표체적작용피길항,기표체수평현저강저.진일보통과면역공침정법증실,재GH3세포중,CaBP-9k여ERα능구직접결합,E2능구상조2자적상호작용.결론 자격소가능통과ERα도경유도GH3세포CaBP-9k적표체,차CaBP-9k여ERα능구직접결합상호작용.제시CaBP-9k가능삼여GH3세포ER도경상관적생물학효응.
Objective To detect the effects of 17 β-estradiol(E2)on the expression of Calbindin-D9k (CaBP-9k) in pituitary GH3 cells,and to investigate the antagonistic effect of a selective estrogen receptor antagonist,ⅡCI 182780 on CaBP-9k expression.Methods A rat pituitary prolactinoma cell line,GH3 cell was used as the in vitro model.The localization of CaBP-9k in GH3 cells was observed by immunofluorescence.GH3 cells were cultured with exogenous E2-added medium for 24 hours,and the concentrations of E2 were 10-8,10-9,10-10M,respectively.mRNA and protein expression levels of CaBP-9k in different groups were analyzed by RT-PCR and Western blot analysis.The estrogen receptor antagonist,and ⅡCI 182780 was added to GH3 cells before E2 administration (10-8M)with the concentration of 10-6M,in order to investigate the regulation of ER-mediated pathway on the expression of CaBP-9k.Immunoprecipitation was used to detect the interaction between CaBP-9k and ERα.Results E2 had significant stimulatory effect on the CaBP-9k expression of GH3 cells in a dose dependent manner,and the expression level of CaBP-9k was higher when treated with a higher concentration of E2.ⅡCI 182780 could suppress the stimulatory effect of E2 on the CaBP-9k expression of GH3 cells.The expression level of CaBP-9k was significantly reduced by coadministration of E2 with ⅡCI 182780 in GH3 cells,which meant the CaBP-9k expression was mediated through ERα pathway.The immunoprecipitation results further illustrated the fact that CaBP-9k could directly interact with ERα,and E2 could increase the interaction between CaBP-9k and ERα.Conclusion Estrogen might induce CaBP-9k expression via ERα mediated pathway and CaBP-9k could directly combine with ERα,suggesting that CaBP-9k might be involved in the biological effects mediated by ER pathway in GH3 cells.