摘要
目的蜕膜化催乳素在妊娠的建立与维持中具有重要作用,但目前对参与蜕膜化催乳素表达调节因子的研究较少。文章旨在探讨孤儿核受体Nur77在人子宫内膜间质细胞蜕膜化过程中对蜕膜化标志基因(Prolactin,PRL)的作用。方法制备Ad-Flag-Nur77重组腺病毒,通过荧光素酶报告基因和荧光定量PCR结合重组腺病毒介导的Nur77过表达及小干扰RNA沉默Nur77基因的实验,阐述Nur77对蜕膜催乳素的调控作用。结果成功获得滴度为8×1010ifu/ml的重组Nur77腺病毒,该腺病毒可在子宫内膜间质细胞中高表达Flag-Nur77融合蛋白;体外培养的人子宫内膜间质细胞经8-Br-cAMP和甲羟孕酮(medroxy progesterone,MPA)刺激后,Nur77的表达明显增加,并在体外诱导蜕膜化培养的第3天达到高峰;过表达Nur77可明显上调人子宫内膜间质细胞中dPRL(-332/+65)启动子活性>4倍,并以浓度依赖的方式明显增加人子宫内膜间质细胞中PRL mRNA的表达,P<0.01;功能缺失实验进一步表明基因沉默内源性Nur77蛋白表达明显抑制dPRL启动子活性达60%,同时显著抑制8-Br-cAMP和MPA诱导的人子宫内膜间质细胞中PRLmRNA的表达,P<0.01。结论孤儿核受体Nur77参与8-Br-cAMP和MPA诱导子宫内膜间质细胞蜕膜化过程中PRL的表达调控。
Objective The research on the importance of prolactin（PRL） in the establishment and maintenance of pregnancy is insufficient which is about the factors involved in the regulation of decidual PRL expression.This study aimed to explore the functional role of the orphan nuclear receptor Nur77 on the decidual PRL gene expression in human endometrial stromal cells（hESCs）.Methods We constructed adenovirus-Flag-Nur77 to infect human endometrial stromal cells.Through experiments on the recombinant adenovirus-mediated overexpression of Nur77 and siRNA knockdown of the Nur77 gene,we studied the regulatory effect of Nur77 on the expression of decidual PRL during the decidualization process using quantitative real-time PCR and luciferase reporter assay.Results We successfully obtained recombinant adenovirus-Flag-Nur77 of 8×1010 ifu/mL,which highly expressed the Flag-Nur77 fusion protein in hESCs.The expression of Nur77 mRNA was significantly upregulated after stimulation with 8Br-cAMP and MPA,reaching the peak at 3 days.The adenovirus-mediated overexpression of Nur77 enhanced the decidual PRL promoter（dPRL/-332Luc） activity over 4 times and markedly increased the PRL mRNA expression in hESCs in a concentration-dependent manner（P0.01）.Furthermore,Nur77 knockdown decreased the decidual PRL promoter activity by 60% and substantially inhibited the PRL mRNA expression induced by cAMP/MPA in hESCs（P0.01）.Conclusion The orphan nuclear receptor Nur77 is a novel transcription factor involved in the regulation of the expression of the PRL gene during the decidualization of hESCs induced by 8-Br-cAMP and MPA.
出处
《医学研究生学报》
CAS
2011年第1期5-10,共6页
Journal of Medical Postgraduates
基金
国家自然科学基金(30900727)
国家重点基础研究发展计划(2010CB945104)
江苏省自然科学基金(BK2009038)
南京市卫生局资金(QYK09172)资助项目
留学回国人员科技活动择优资助项目
