本文選題：PGR　切入點：ADAMTS-1　出處：《重慶大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：孕酮在哺乳動物雌性生殖活動中發(fā)揮了非常重要的作用，孕酮的生理功能主要是通過孕酮受體（Progesterone receptor, PGR）介導(dǎo)的。孕酮受體基因敲除小鼠模型已經(jīng)證實PGR基因?qū)τ诓溉閯游锎菩陨彻δ艿闹匾�，尤其在排卵中發(fā)揮了不可替代的作用。近年來，通過基因芯片實驗已經(jīng)篩選出了大量PGR的下游基因，但至今未能在這些下游基因的啟動子區(qū)域上找到傳統(tǒng)意義上的孕酮響應(yīng)元件。ADAMTS-1和Cathepsin L曾被認為是PGR基因在卵巢中介導(dǎo)卵泡壁破裂的關(guān)鍵信號分子，但這兩種蛋白如何受PGR基因的調(diào)控以及其在卵泡壁破裂中發(fā)揮了什么樣的作用，一直還不清楚。 本實驗以性未成熟的雌性昆明小鼠為研究對象，用外源促性腺激素（PMSG/hCG）誘導(dǎo)小鼠超排卵，用實時熒光定量PCR研究PGR、ADAMTS-1和Cathepsin L mRNA在圍排卵期卵巢中的表達變化，發(fā)現(xiàn)ADAMTS-1mRNA表達量在PMSG注射后24h開始有顯著增高，一直持續(xù)到hCG注射后4h，在hCG注射后12h又進一步有顯著性的增高，而hCG注射后12h正好是小鼠的排卵時刻，這說明ADAMTS-1不僅在卵泡發(fā)育過程中有重要作用，更在排卵過程中發(fā)揮了關(guān)鍵作用；并且，ADAMTS-1mRNA表達量在小鼠排卵時刻的進一步增高是發(fā)生在PGRmRNA表達量達到最大值之后，這說明很可能是PGR誘導(dǎo)了ADAMTS-1在小鼠排卵時刻表達量的突然性增高。相反，在整個外源促性腺激素處理小鼠的過程中，卵巢中Cathepsin L mRNA表達量一直沒有顯著變化（P0.05），說明Cathepsin LmRNA在卵巢中主要以組成性表達為主，外源促性腺激素并不能引起Cathepsin LmRNA在整個卵巢水平的變化。進一步，用孕酮受體拮抗劑RU486來處理小鼠，在hCG注射后12h檢測卵巢中基因mRNA的表達，發(fā)現(xiàn)RU486能顯著抑制外源促性腺激素對ADAMTS-1表達的刺激作用（P 0.05），這說明外源促性腺激素對ADAMTS-1mRNA在小鼠排卵時刻的刺激是由PGR介導(dǎo)的。 此外，低氧誘導(dǎo)因子1α（Hypoxia inducible factor1, HIF-1α）和HIF-1β作為已知的在小鼠卵巢中受PGR調(diào)節(jié)的下游基因，它們以異源二聚體的形式（即HIF-1）來發(fā)揮轉(zhuǎn)錄因子的功能。而且，在人血管內(nèi)皮細胞中已經(jīng)發(fā)現(xiàn)HIF-1能直接結(jié)合到ADAMTS-1啟動子區(qū)域。因此，我們自然而然地假設(shè)，在小鼠卵巢中，PGR對是通過HIF-1來間接調(diào)控ADAMTS-1表達的。為了驗證這個假設(shè)，我們以方便研究且同時表達PGR、ADAMTS-1、HIF-1α和HIF-1β基因的人乳腺癌細胞系（MCF-7）為研究對象，用不同濃度（10nM、100nM和1μM）的外源孕激素醋酸甲羥孕酮（MPA）處理MCF-7細胞，發(fā)現(xiàn)隨著MPA濃度的升高，MPA對ADAMTS-1mRNA表達的刺激越來越明顯（P 0.05），，對HIF-1α和HIF-1β及其典型的下游靶基因VEGFA mRNA的表達雖然也有逐步升高的趨勢，但差異并不顯著（P0.05）。在此基礎(chǔ)上，用不同濃度RU486（0.1μM、1μM和10μM）處理細胞，發(fā)現(xiàn)隨著濃度的升高，RU486對ADAMTS-1mRNA表達抑制越來越明顯；對HIF-1α、HIF-1β和VEGFA卻沒有抑制作用；并且，在RU486濃度為10μM時，VEGFA mRNA的表達量反而有極顯著性的升高（P 0.01）。這些結(jié)果表明，在MCF-7細胞中，PGR同樣能夠調(diào)節(jié)ADAMTS-1的表達，但對HIF-1α、HIF-1β和VEGFA卻沒有明顯的調(diào)節(jié)作用。用氯化鈷來模擬低氧環(huán)境并刺激細胞中HIF-1α蛋白質(zhì)濃度的升高，發(fā)現(xiàn)氯化鈷處理能同時刺激ADAMTS-1mRNA和蛋白質(zhì)的表達，這說明在MCF-7細胞中，HIF-1同樣能調(diào)節(jié)ADAMTS-1的表達。然而，用MPA和氯化鈷同時處理MCF-7細胞，HIF-1α和HIF-1β mRNA無明顯變化，但VEGFA mRNA卻有顯著的升高（P 0.05），而PGR mRNA表達量被顯著抑制（P 0.05），同時ADAMTS-1mRNA也有明顯的抑制趨勢。這說明在MCF-7細胞中，ADAMTS-1的表達主要受PGR的調(diào)控，而高濃度的HIF-1α蛋白質(zhì)卻能反過來抑制PGR的表達，并進一步抑制ADAMTS-1的表達。因此，在MCF-7細胞中，PGR對ADAMTS-1表達的調(diào)控可能不是由HIF-1介導(dǎo)的，至少不完全是。 綜上，在小鼠卵巢中，PGR介導(dǎo)了外源促性腺激素對小鼠排卵時刻ADAMTS-1mRNA表達的刺激，但外源促性腺激素并不影響小鼠卵巢中Cathepsin L mRNA的表達。進一步，在MCF-7細胞中，PGR同樣能調(diào)控ADAMTS-1的表達，但對HIF-1α和HIF-1β這兩個已知的在小鼠卵巢中受PGR調(diào)節(jié)的下游基因無明顯的調(diào)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.

【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R714.8

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相關(guān)期刊論文 前2條

1 羅倩萍;吳艷青;張正紅;王正朝;;顆粒細胞在哺乳動物卵巢排卵過程中的調(diào)控作用[J];生命科學(xué);2013年06期

2 張寶云;狄冉;儲明星;王憑青;魯浪;;孕酮受體基因的研究進展[J];遺傳;2008年12期

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