孕酮受体基因（PGR）介导小鼠排卵的关键信号分子研究

发布时间：2018-03-15 08:08

本文选题：PGR　切入点：ADAMTS-1　出处：《重庆大学》2014年硕士论文　论文类型：学位论文

【摘要】：孕酮在哺乳动物雌性生殖活动中发挥了非常重要的作用，孕酮的生理功能主要是通过孕酮受体（Progesterone receptor, PGR）介导的。孕酮受体基因敲除小鼠模型已经证实PGR基因对于哺乳动物雌性生殖功能的重要性，尤其在排卵中发挥了不可替代的作用。近年来，通过基因芯片实验已经筛选出了大量PGR的下游基因，但至今未能在这些下游基因的启动子区域上找到传统意义上的孕酮响应元件。ADAMTS-1和Cathepsin L曾被认为是PGR基因在卵巢中介导卵泡壁破裂的关键信号分子，但这两种蛋白如何受PGR基因的调控以及其在卵泡壁破裂中发挥了什么样的作用，一直还不清楚。本实验以性未成熟的雌性昆明小鼠为研究对象，用外源促性腺激素（PMSG/hCG）诱导小鼠超排卵，用实时荧光定量PCR研究PGR、ADAMTS-1和Cathepsin L mRNA在围排卵期卵巢中的表达变化，发现ADAMTS-1mRNA表达量在PMSG注射后24h开始有显著增高，一直持续到hCG注射后4h，在hCG注射后12h又进一步有显著性的增高，而hCG注射后12h正好是小鼠的排卵时刻，这说明ADAMTS-1不仅在卵泡发育过程中有重要作用，更在排卵过程中发挥了关键作用；并且，ADAMTS-1mRNA表达量在小鼠排卵时刻的进一步增高是发生在PGRmRNA表达量达到最大值之后，这说明很可能是PGR诱导了ADAMTS-1在小鼠排卵时刻表达量的突然性增高。相反，在整个外源促性腺激素处理小鼠的过程中，卵巢中Cathepsin L mRNA表达量一直没有显著变化（P0.05），说明Cathepsin LmRNA在卵巢中主要以组成性表达为主，外源促性腺激素并不能引起Cathepsin LmRNA在整个卵巢水平的变化。进一步，用孕酮受体拮抗剂RU486来处理小鼠，在hCG注射后12h检测卵巢中基因mRNA的表达，发现RU486能显著抑制外源促性腺激素对ADAMTS-1表达的刺激作用（P 0.05），这说明外源促性腺激素对ADAMTS-1mRNA在小鼠排卵时刻的刺激是由PGR介导的。此外，低氧诱导因子1α（Hypoxia inducible factor1, HIF-1α）和HIF-1β作为已知的在小鼠卵巢中受PGR调节的下游基因，它们以异源二聚体的形式（即HIF-1）来发挥转录因子的功能。而且，在人血管内皮细胞中已经发现HIF-1能直接结合到ADAMTS-1启动子区域。因此，我们自然而然地假设，在小鼠卵巢中，PGR对是通过HIF-1来间接调控ADAMTS-1表达的。为了验证这个假设，我们以方便研究且同时表达PGR、ADAMTS-1、HIF-1α和HIF-1β基因的人乳腺癌细胞系（MCF-7）为研究对象，用不同浓度（10nM、100nM和1μM）的外源孕激素醋酸甲羟孕酮（MPA）处理MCF-7细胞，发现随着MPA浓度的升高，MPA对ADAMTS-1mRNA表达的刺激越来越明显（P 0.05），，对HIF-1α和HIF-1β及其典型的下游靶基因VEGFA mRNA的表达虽然也有逐步升高的趋势，但差异并不显著（P0.05）。在此基础上，用不同浓度RU486（0.1μM、1μM和10μM）处理细胞，发现随着浓度的升高，RU486对ADAMTS-1mRNA表达抑制越来越明显；对HIF-1α、HIF-1β和VEGFA却没有抑制作用；并且，在RU486浓度为10μM时，VEGFA mRNA的表达量反而有极显著性的升高（P 0.01）。这些结果表明，在MCF-7细胞中，PGR同样能够调节ADAMTS-1的表达，但对HIF-1α、HIF-1β和VEGFA却没有明显的调节作用。用氯化钴来模拟低氧环境并刺激细胞中HIF-1α蛋白质浓度的升高，发现氯化钴处理能同时刺激ADAMTS-1mRNA和蛋白质的表达，这说明在MCF-7细胞中，HIF-1同样能调节ADAMTS-1的表达。然而，用MPA和氯化钴同时处理MCF-7细胞，HIF-1α和HIF-1β mRNA无明显变化，但VEGFA mRNA却有显著的升高（P 0.05），而PGR mRNA表达量被显著抑制（P 0.05），同时ADAMTS-1mRNA也有明显的抑制趋势。这说明在MCF-7细胞中，ADAMTS-1的表达主要受PGR的调控，而高浓度的HIF-1α蛋白质却能反过来抑制PGR的表达，并进一步抑制ADAMTS-1的表达。因此，在MCF-7细胞中，PGR对ADAMTS-1表达的调控可能不是由HIF-1介导的，至少不完全是。综上，在小鼠卵巢中，PGR介导了外源促性腺激素对小鼠排卵时刻ADAMTS-1mRNA表达的刺激，但外源促性腺激素并不影响小鼠卵巢中Cathepsin L mRNA的表达。进一步，在MCF-7细胞中，PGR同样能调控ADAMTS-1的表达，但对HIF-1α和HIF-1β这两个已知的在小鼠卵巢中受PGR调节的下游基因无明显的调控作用。
[Abstract]:Progesterone plays a very important role in female reproductive activity in mammals, the physiological function of progesterone is mainly by progesterone receptor (Progesterone receptor PGR) mediated by progesterone receptor gene knockout mice have confirmed the importance of PGR gene to mammalian female reproductive function, especially played an irreplaceable role in ovulation. In recent years, through microarray experiments have been screened out downstream genes in a large number of PGR, but has not been in these downstream gene promoter region found in the traditional sense of the progesterone response element.ADAMTS-1 and Cathepsin L was thought to be mediated PGR gene in ovarian follicle wall rupture of key signaling molecules, but how these two protein regulated by PGR gene and the follicle wall rupture plays a role in what has been, is not clear.
In this experiment, immature female Kunming mice as the research object, using exogenous gonadotropin (PMSG/hCG) induced by superovulation, using real-time quantitative PCR study of PGR, the expression of ADAMTS-1 and Cathepsin L mRNA in the peri ovulatory period in the ovary, the expression of ADAMTS-1mRNA was at PMSG after injection of 24h has increased significantly, has been until after the injection of hCG 4H in hCG 12h after injection and further increased significantly, and hCG after injection of 12h is in ovulation time, indicating that ADAMTS-1 not only plays an important role in follicular development process, it has played a key role in the ovulation process; and the expression of ADAMTS-1mRNA in mouse ovulation time the increase is reached the maximum expression after PGRmRNA, indicating that PGR may be induced the expression of ADAMTS-1 in mouse sudden increase ovulation time. In contrast, in the whole To promote the process of exogenous hormone treatments in mice, ovarian volume there have been no significant changes in the expression of mRNA Cathepsin L (P0.05), Cathepsin LmRNA in ovary mainly expressed constitutively, exogenous gonadotropin is not caused by Cathepsin LmRNA in the changes in the ovarian levels. Further, a progesterone receptor antagonist RU486 treated mice, 12h expression in ovarian detection after hCG injection in mRNA gene, found that stimulation of RU486 can significantly inhibit the exogenous gonadotropin on the expression of ADAMTS-1 (P 0.05), indicating that exogenous gonadotropin on ADAMTS-1mRNA in mice the time of ovulation stimulation is mediated by PGR.
In addition, hypoxia inducible factor 1 (Hypoxia inducible factor1, HIF-1 alpha and HIF-1 beta) as known by regulating downstream gene PGR in mouse ovary, with two heterologous dimer form (HIF-1) to play the function of transcription factors. Moreover, in human vascular endothelial cells have been found in HIF-1 direct binding to the promoter region of ADAMTS-1. Therefore, we naturally assume that in mouse ovary, PGR is used by HIF-1 to indirectly regulate the expression of ADAMTS-1. In order to test this hypothesis, we study and at the same time to facilitate the expression of PGR, ADAMTS-1, human breast cancer cell line gene HIF-1 alpha and HIF-1 beta (MCF-7) for the object of study, with different concentrations (10nM, 100nM and 1 M) exogenous progesterone medroxyprogesterone acetate (MPA) treatment of MCF-7 cells, found that with the increasing of MPA concentration, MPA expression of ADAMTS-1mRNA stimulation is more and more obvious (P 0.05), HIF-1 alpha and HI The expression of F-1 beta and its downstream target gene VEGFA typical mRNA although there are gradually rising trend, but the difference was not significant (P0.05). On this basis, with different concentrations of RU486 (0.1 M, 1 M and 10 M) treated cells, found that with the increase of concentration, the inhibition of RU486 gene expression is more and more obvious for ADAMTS-1mRNA; on HIF-1 alpha, HIF-1 beta and VEGFA had no inhibitory effect; and, when the RU486 concentration is 10 M, the expression of VEGFA mRNA but increased significant (P 0.01). These results suggest that in MCF-7 cells, PGR can regulate the expression of ADAMTS-1, but for HIF-1 alpha, beta HIF-1 and VEGFA but no obvious regulation effect. With cobalt chloride to simulate hypoxia and increase the concentration of HIF-1 protein in stimulator cells, found that the cobalt chloride treatment can also stimulate the expression of ADAMTS-1mRNA and protein, indicating that in MCF-7 cells, HIF-1 can also regulate ADAM The expression of TS-1. However, at the same time, MCF-7 cells were treated with MPA and cobalt chloride, HIF-1 alpha and HIF-1 beta mRNA had no obvious change, but VEGFA mRNA was increased significantly (P 0.05), while PGR mRNA expression was significantly inhibited (P 0.05), at the same time, ADAMTS-1mRNA also decreased obviously. This shows that in the MCF-7 cells, regulate the expression of ADAMTS-1 is mainly controlled by PGR, and HIF-1 protein in high concentration can inhibit the expression of PGR expression in turn, and further inhibit ADAMTS-1. Therefore, in MCF-7 cells, the regulation of PGR on the expression of ADAMTS-1 may not be mediated by HIF-1, at least not completely.
In conclusion, in mouse ovary, PGR mediated exogenous gonadotropin on ovulation in mice when the stimulation of ADAMTS-1mRNA expression, but exogenous gonadotropin does not affect the expression of Cathepsin in mouse ovary L mRNA. Further, in MCF-7 cells, PGR can also regulate the expression of ADAMTS-1, but the HIF-1 alpha and HIF-1 beta this two known downstream genes in mouse ovary by regulating PGR without obvious regulation.

【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

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