骨形态发生蛋白-7（BMP-7）在小鼠蜕膜细胞的表达及功能研究
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摘要
目的:胚胎着床是胎生哺乳动物及人类生殖生理的重要环节,是母体子宫与胚胎同步发育、在特定时期即“着床窗”开放期间精确调节、快速完成的生理现象。妊娠需要子宫组织发生广泛的重建,子宫内膜细胞生长和分化,即发生蜕膜化。子宫内膜的蜕膜化过程对于成功的着床和妊娠的维持是必需的。蜕膜化过程涉及黏附分子、蛋白水解酶、生长因子、细胞因子、血管活性因子以及多种基因的相互作用和调节。已有研究表明BMPs家族在小鼠胚胎发育过程中都有表达,为进一步研究其家族中BMP-7在小鼠子宫中的表达规律和作用,本实验采用细胞原代培养、荧光定量PCR、免疫组织化学、免疫细胞化学、Western blotting等方法从mRNA和蛋白水平检测BMP-7在妊娠小鼠子宫中的表达,探索其在子宫内膜蜕膜化过程中的作用。
方法: 1.RT-PCR和免疫组织化学技术分别观察BMP-7的mRNA和蛋白在妊娠第5-8日小鼠子宫中的表达情况;2.原代培养小鼠子宫基质细胞,用孕酮、雌二醇和cAMP诱导蜕膜化,并在诱导蜕膜化的同时分别加入1.0ng/ml、10ng/ml、100ng/ml BMP-7重组腺病毒,于24h、48h、72h、96h检测催乳素的表达;3.去卵巢小鼠随机分3组,分别皮下注射芝麻油(0.1ml/只)、孕酮(2.0mg/只)、RU486(2.0mg/只)+孕酮(2.0mg/只),于注射后0h、6h、12h、24h处死小鼠收集子宫,提取子宫RNA和总蛋白,荧光定量PCR、Western blotting检测BMP-7的表达情况。
结果:1.妊娠第5-8日,BMP-7 mRNA在小鼠子宫着床位点的表达高于着床旁组织(P<0.01),且在着床点的表达随着妊娠天数的增加逐渐增强(P<0.05)。妊娠第5日、第6日,BMP-7蛋白分别表达于植入胚胎周围的基质细胞和初级蜕膜带;第7日、第8日,主要表达于次级蜕膜带以及植入位点系膜侧基质细胞。2.在小鼠子宫基质细胞的蜕膜化过程中,BMP-7重组腺病毒组催乳素的mRNA和蛋白表达均显著增加(P<0.01),其中10ng/ml组增加最明显(P<0.01)。3.去卵巢小鼠模型研究显示:孕酮上调了小鼠子宫组织BMP-7的表达(P<0.01),孕酮受体拮抗剂RU486能有效阻断孕酮对BMP-7的上调作用(P<0.05)
结论:BMP-7基因在小鼠子宫内的表达受孕酮调控,具有促进小鼠子宫内膜蜕膜化的效应。
Objectives: Embryo implantation is an important and complicated physiological process in viviparous mammalian and human reproduction, which is also a precisely regulated and quickly accomplished physical phenomenon synchronized with development of maternal uterus and embryo at a particular time named "implantation window". Pregnancy needs extensive reconstruction of uterus as well as growth and differentiation of endometrial cell, which is decidualization. In addition, the decidualization process involves the interaction and regulation of adhesion molecules, proteolytic enzymes, growth factors, cytokines, vasoactive factors and multiple genes. Comparative research shows BMPs were expressed during embryo development in mice. To further study the expression pattern and roles of BMP-7 in uterus, we use various approaches including primary cell culture, quantitative PCR, immunohistochemistry, immunocytochemistry, western blotting. We detect the levels of BMP-7 mRNA and protein in the pregnant mouse uterine endometria in order to explore the role of BMP-7 in decidualization process.
Methods: 1. RT-PCR and immunohistochemistry were applied to analyze BMP-7 mRNA and protein in mouse uterine endometrium from day 5 to 8 of pregnancy. 2. Mouse uterine stromal cells were isolated from uterus of pregnant day 4 and cultured in DMEM containing 10% fetal bovine serum. Decidualization of stromal cells was induced using progesterone, estradiol, and cAMP followed by transfected with 1.0 ng/ml, 10 ng/ml, 100 ng/ml recombinant BMP-7 adenovirus, respectively. After 72 h of culture, the expression of prolactin, a typical marker of decidual cells, was detected by RT-PCR and Western blotting. 3. Ovariectomized mice were randomly divided into 3 groups. Each group were subcutaneously injected with sesame oil (0.1ml/mouse), progesterone (2mg/mouse), RU486 (2mg/mouse) and progesterone (2mg/mouse), respectively. All mice were killed at 0h, 6h, 12h, 24h after injection to collect the utera samples in order to achieve uterine RNA and total protein from which BMP-7 levels was later examined by quantitative PCR and Western blotting.
Results: 1. The expression level of BMP-7 mRNA was higher in the implantation sites than that in inter-implantation sites during day 5 to 8 of pregnancy (P<0.01), and the level increased gradually as days passed by in the implantation sites (P<0.05). BMP-7 protein was expressed in the decidualizing stromal cells tightly surrounding the blastocyst at day 5 and in the primary decidual zone at day 6. Progessively, the positive detection appeared mainly in the secondary decidual zone especially in the stromal cells of mesometrial pole at day 7 and day 8. 2. During decidualization of stromal cells, the expression of prolactin protein in recombinant BMP-7 adenovirus group were significantly increased (P<0.01), especially in 10 ng/ml group (P<0.01). 3. Progesterone was demonstrated with ovariectomized mouse models to up-regulate the expression of BMP-7 mRNA (P<0.01). RU486, an specific antagonist of progesterone receptor, is capable of abrogating progesterone effect on BMP-7.
Conclusions: BMP-7 gene was demonstrated to be involed in inducing the decidualization of uterine stromal cells in early pregnant mice and this effect was regulated by progesterone.
方法: 1.RT-PCR和免疫组织化学技术分别观察BMP-7的mRNA和蛋白在妊娠第5-8日小鼠子宫中的表达情况;2.原代培养小鼠子宫基质细胞,用孕酮、雌二醇和cAMP诱导蜕膜化,并在诱导蜕膜化的同时分别加入1.0ng/ml、10ng/ml、100ng/ml BMP-7重组腺病毒,于24h、48h、72h、96h检测催乳素的表达;3.去卵巢小鼠随机分3组,分别皮下注射芝麻油(0.1ml/只)、孕酮(2.0mg/只)、RU486(2.0mg/只)+孕酮(2.0mg/只),于注射后0h、6h、12h、24h处死小鼠收集子宫,提取子宫RNA和总蛋白,荧光定量PCR、Western blotting检测BMP-7的表达情况。
结果:1.妊娠第5-8日,BMP-7 mRNA在小鼠子宫着床位点的表达高于着床旁组织(P<0.01),且在着床点的表达随着妊娠天数的增加逐渐增强(P<0.05)。妊娠第5日、第6日,BMP-7蛋白分别表达于植入胚胎周围的基质细胞和初级蜕膜带;第7日、第8日,主要表达于次级蜕膜带以及植入位点系膜侧基质细胞。2.在小鼠子宫基质细胞的蜕膜化过程中,BMP-7重组腺病毒组催乳素的mRNA和蛋白表达均显著增加(P<0.01),其中10ng/ml组增加最明显(P<0.01)。3.去卵巢小鼠模型研究显示:孕酮上调了小鼠子宫组织BMP-7的表达(P<0.01),孕酮受体拮抗剂RU486能有效阻断孕酮对BMP-7的上调作用(P<0.05)
结论:BMP-7基因在小鼠子宫内的表达受孕酮调控,具有促进小鼠子宫内膜蜕膜化的效应。
Objectives: Embryo implantation is an important and complicated physiological process in viviparous mammalian and human reproduction, which is also a precisely regulated and quickly accomplished physical phenomenon synchronized with development of maternal uterus and embryo at a particular time named "implantation window". Pregnancy needs extensive reconstruction of uterus as well as growth and differentiation of endometrial cell, which is decidualization. In addition, the decidualization process involves the interaction and regulation of adhesion molecules, proteolytic enzymes, growth factors, cytokines, vasoactive factors and multiple genes. Comparative research shows BMPs were expressed during embryo development in mice. To further study the expression pattern and roles of BMP-7 in uterus, we use various approaches including primary cell culture, quantitative PCR, immunohistochemistry, immunocytochemistry, western blotting. We detect the levels of BMP-7 mRNA and protein in the pregnant mouse uterine endometria in order to explore the role of BMP-7 in decidualization process.
Methods: 1. RT-PCR and immunohistochemistry were applied to analyze BMP-7 mRNA and protein in mouse uterine endometrium from day 5 to 8 of pregnancy. 2. Mouse uterine stromal cells were isolated from uterus of pregnant day 4 and cultured in DMEM containing 10% fetal bovine serum. Decidualization of stromal cells was induced using progesterone, estradiol, and cAMP followed by transfected with 1.0 ng/ml, 10 ng/ml, 100 ng/ml recombinant BMP-7 adenovirus, respectively. After 72 h of culture, the expression of prolactin, a typical marker of decidual cells, was detected by RT-PCR and Western blotting. 3. Ovariectomized mice were randomly divided into 3 groups. Each group were subcutaneously injected with sesame oil (0.1ml/mouse), progesterone (2mg/mouse), RU486 (2mg/mouse) and progesterone (2mg/mouse), respectively. All mice were killed at 0h, 6h, 12h, 24h after injection to collect the utera samples in order to achieve uterine RNA and total protein from which BMP-7 levels was later examined by quantitative PCR and Western blotting.
Results: 1. The expression level of BMP-7 mRNA was higher in the implantation sites than that in inter-implantation sites during day 5 to 8 of pregnancy (P<0.01), and the level increased gradually as days passed by in the implantation sites (P<0.05). BMP-7 protein was expressed in the decidualizing stromal cells tightly surrounding the blastocyst at day 5 and in the primary decidual zone at day 6. Progessively, the positive detection appeared mainly in the secondary decidual zone especially in the stromal cells of mesometrial pole at day 7 and day 8. 2. During decidualization of stromal cells, the expression of prolactin protein in recombinant BMP-7 adenovirus group were significantly increased (P<0.01), especially in 10 ng/ml group (P<0.01). 3. Progesterone was demonstrated with ovariectomized mouse models to up-regulate the expression of BMP-7 mRNA (P<0.01). RU486, an specific antagonist of progesterone receptor, is capable of abrogating progesterone effect on BMP-7.
Conclusions: BMP-7 gene was demonstrated to be involed in inducing the decidualization of uterine stromal cells in early pregnant mice and this effect was regulated by progesterone.
引文
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