摘要
目的人子宫内膜间质细胞蜕膜化标志蛋白胰岛素样生长因子结合蛋白-1(insulin-like growth factor binding protein-1,IGFBP-1)参与调控月经周期、排卵、蜕膜化、胚胎种植以及胎儿生长等生理过程,本研究旨在探索锌指蛋白KLF12对人子宫内膜间质细胞体外蜕膜化过程中IGFBP-1表达的影响。方法制备Ad-Flag-KLF12重组腺病毒,通过荧光定量聚合酶链式反应(PCR)和酶联免疫吸附试验(ELISA)结合重组腺病毒介导的KLF12过表达实验,阐述人子宫内膜间质细胞体外蜕膜化过程中KLF12对IGFBP-1表达调控作用。结果成功获得滴度为2×1011ifu/ml的重组KLF12腺病毒,该腺病毒可在人子宫内膜间质细胞中高表达Flag-KLF12融合蛋白;体外分离培养的人子宫内膜间质细胞经8-Br-cAMP和醋酸甲羟孕酮(medroxy progesterone acetate,MPA)联合诱导蜕膜化后,KLF12的mRNA表达随着诱导时间延长逐渐降低,72h降低最明显,较未刺激降低约70%;过表达KLF12显著抑制8-Br-cAMP和MPA诱导的人子宫内膜间质细胞中IGFBP-1mRNA的表达,并以时间依赖的方式明显抑制IGFBP-1的分泌(P<0.01);过表达KLF12明显抑制人子宫内膜间质细胞体外蜕膜化过程中IGFBP-1启动子转录活性。结论锌指蛋白KLF12抑制人子宫内膜间质细胞体外蜕膜化过程中IGFBP-1的表达与分泌。
Objective： Insulin-like growth factor-1 （IGFBP-1）, one of marker proteins expressed during endometrial decidualization, is particularly important in human female reproductive physiology, which regulates menstrual cycles, puberty, ovulation, decidualization, implantation ＇and fetal growth. Members of the KriJppel-like factors（KLFs）family of transcription factors play the roles in maternal endometrium development during embryo implantation. However the specific role of Krtippel-like factor 12（KI.F12）in endometrium development has not yet been described. This study is aimed to explore the functional role of the KLF12 on the expression of IGFBP-1 in human endometrial stromal cells（hESCs）during ttecidualization in vitro. Methods. Adenovirus-Flag-KLF12 was constructed and infected to human endometrial stromal cells. Through the recombinant adenovirus mediated overexpression of KLF12, the regulatory effect of KLF12 on the exPression of IGFBP-1 in hESCs during decidualization in vitro was studied by quantitative real-time PCR and enzyme iinked immumosorbent assays（ELISA）. Results. The recombinant adenovirus-Flag-KLF12 of 2 × 10^11 ifu/ml was successfully obtained,which highly expressed Flag-KLF12 fusion protein in hESCs. The expression of KLFi2 mRNA was significantly decreased at 72 hours by approximately 70 % after Stimulation with 8-Br-cAMP and medroxyprogesterone acetate（MPA） to＇induce decidualization in vitro. ＂The adenovirus-mediated overexpression of KLF12 markedly suppressed IGFBP-1 mRNA expression by approximately 48% and inhibited the secretion in a time-dependent manner （P〈0.01）in hESCs during decidualization in vitro. Moreover, luciferase reporter assays demonstrated that overexpression of KLF12 deregulated IGFBP-1 promoter activity in hESCs during decidualization in vitro. Conclusions. KLF12 negatively regulates the expression of IGFBP-1 in hESCs during decidualization in vitro.
出处
《生殖医学杂志》
CAS
2014年第5期393-400,共8页
Journal of Reproductive Medicine
基金
国家自然科学基金(81070492
81170570
81370683)
"六大人才高峰"项目基金(2012-WSN-005)
南京市卫生局基金(YKK12054)
留学回国人员科研启动基金(2011-1139)资助
