本文關(guān)鍵詞：蛋白激酶MAP3K8對(duì)小鼠黃體孕酮合成的調(diào)節(jié)機(jī)理研究　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： MAP3K-8 雌二醇 雌二醇膜受體 黃體 孕酮

【摘要】：黃體是卵泡排卵后形成的一個(gè)短暫性的分泌器官,在調(diào)節(jié)發(fā)情周期及妊娠維持過(guò)程中發(fā)揮重要的作用。黃體的主要功能是通過(guò)合成及分泌孕酮調(diào)節(jié)多種生理活動(dòng),而黃體中孕酮的合成受催乳素、促黃體素、雌二醇、前列腺素等激素的調(diào)節(jié)。在嚙齒類(lèi)動(dòng)物中,催乳素和雌二醇是促進(jìn)孕酮合成的主要激素,促黃體素在小鼠黃體中調(diào)節(jié)孕酮合成的作用較小。大量的研究證明了雌二醇在調(diào)節(jié)黃體孕酮合成的過(guò)程中起著非常關(guān)鍵的作用,但對(duì)其作用機(jī)理仍不清楚。本實(shí)驗(yàn)室之前的研究發(fā)現(xiàn)MAP3K8與激素的合成與分泌有關(guān)。因此,本研究的主要目標(biāo)是研究MAP3K8在小鼠黃體中的表達(dá),并進(jìn)一步的探索該基因?qū)τ谠型铣傻恼{(diào)節(jié)作用及機(jī)理。首先利用免疫組織化學(xué)(IHC)方法檢測(cè)了MAP3K8在小鼠卵巢中的表達(dá),結(jié)果顯示MAP3K8特異性的在卵巢黃體細(xì)胞表達(dá),顆粒細(xì)胞及膜細(xì)胞中未檢測(cè)到陽(yáng)性信號(hào)。RT-qPCR及WB方法對(duì)不同時(shí)期的黃體中MAP3K8表達(dá)的分析結(jié)果證明MAP3K8在中期黃體中的表達(dá)量顯著高于早期及晚期黃體。為了進(jìn)一步探討MAP3K8對(duì)黃體功能的調(diào)節(jié),利用MAP3K8-siRNA及MAP3K8化學(xué)抑制劑(MAP3K8 inhibitor, MAP3K8i)阻斷黃體化顆粒細(xì)胞中MAP3K8信號(hào),結(jié)果發(fā)現(xiàn)黃體化顆粒細(xì)胞中孕酮的分泌水平降低。體內(nèi)注射MAP3K8i的實(shí)驗(yàn)結(jié)果與細(xì)胞培養(yǎng)的結(jié)果一致,即注射MAP3K8i導(dǎo)致小鼠血清及黃體組織中孕酮的含量降低。之后的研究發(fā)現(xiàn)注射MAP3K8i后孕鼠黃體中孕酮水平的變化會(huì)進(jìn)一步的影響胚胎著床。以上實(shí)驗(yàn)結(jié)果證明MAP3K8在調(diào)節(jié)黃體孕酮合成及相應(yīng)的孕酮生理功能中有著重要的作用。此外,本研究發(fā)現(xiàn),雌二醇能夠上調(diào)黃體細(xì)胞中MAP3K8的表達(dá),促黃體素及催乳素均不能調(diào)節(jié)MAP3K8的表達(dá)。MAP3K8-siRNA及MAP3K8i阻斷黃體化顆粒細(xì)胞中MAP3K8信號(hào)后,雌二醇促進(jìn)孕酮合成的功能受到了限制。這些實(shí)驗(yàn)結(jié)果說(shuō)明MAP3K8介導(dǎo)雌二醇對(duì)孕酮合成的調(diào)節(jié)功能。多數(shù)組織中雌二醇通過(guò)其核受體ERa及ERβ發(fā)揮功能,但近期的研究發(fā)現(xiàn)雌二醇也可與其膜受體GPR30結(jié)合發(fā)揮功能。為了證明雌二醇調(diào)節(jié)黃體孕酮合成的信號(hào)通路和相關(guān)的分子機(jī)理,我們首先分析了黃體中雌二醇受體的分子類(lèi)別,結(jié)果表明ERα、ERβ及GPR30均在黃體中表達(dá),并且黃體化的顆粒細(xì)胞中雌二醇能夠快速的調(diào)節(jié)MAP3K8的表達(dá),且這種作用可被膜受體抑制劑G15阻斷,由此證明了雌二醇通過(guò)膜受體GPR30上調(diào)MAP3K8的表達(dá)而發(fā)揮功能。同時(shí)我們的實(shí)驗(yàn)結(jié)果證明MAP3K8對(duì)黃體細(xì)胞中孕酮合成的調(diào)節(jié)是通過(guò)促進(jìn)ERK磷酸化實(shí)現(xiàn)的。綜上所述,MAP3K8在小鼠卵巢的黃體中表達(dá),并且在中期黃體中的表達(dá)量顯著高于早期及晚期黃體。雌二醇通過(guò)膜受體GPR30上調(diào)黃體細(xì)胞中MAP3K8的表達(dá),抑制實(shí)驗(yàn)證明MAP3K8介導(dǎo)了雌二醇促進(jìn)孕酮合成的生理功能。
[Abstract]:The formation of corpus luteum follicle after ovulation a transient organ, in the regulation of the estrous cycle and pregnancy maintenance play an important role in the process. The main function of the corpus luteum is through the synthesis and secretion of progesterone regulates many physiological activities, and luteal progesterone synthesis by prolactin, luteinizing hormone, estradiol, prostaglandins, hormone regulation in rodent animal, prolactin and estradiol promote hormone progesterone synthesis, luteinizing hormone in mice in the regulation of small luteal progesterone synthesis. A large number of studies have proved that estradiol plays a key role in the regulation of luteal progesterone synthesis process, but the mechanism is still unclear. Our previous found MAP3K8 synthesis and secretion of hormones. Therefore, the main goal of this research is to study the expression of MAP3K8 in mouse corpus, and further exploration The gene for regulation of progesterone synthesis and mechanism. Firstly, using immunohistochemical (IHC) method to detect the expression of MAP3K8 in mouse ovary, showed specific expression of MAP3K8 in ovarian granulosa cells and luteal cells, mesangial cells were not detected in the positive signal of.RT-qPCR and the WB method for analysis of MAP3K8 expression in different periods the results show that the expression of MAP3K8 in corpus luteum in mid luteal was significantly higher than that in early and late luteal phase. In order to further investigate the regulation of MAP3K8 on luteal function, using MAP3K8-siRNA and MAP3K8 (MAP3K8 inhibitor, MAP3K8i chemical inhibitors) blocking MAP3K8 signal luteinizing granulosa cells, the results found that the secretion of progesterone level of luteinizing granulosa cells decreased. The experimental results of injection MAP3K8i in vivo and cell culture results, namely MAP3K8i injection of progesterone in serum and luteal tissue in mice The content decreased. Subsequent studies found that affect embryo implantation further changes in luteal progesterone levels in pregnant rats after injection of MAP3K8i. These results demonstrated that MAP3K8 plays an important role in the regulation of luteal progesterone progesterone synthesis and corresponding physiological function. In addition, the study found that estradiol can upregulate the expression of MAP3K8 in luteal cells the luteinizing hormone and prolactin were not the expression of.MAP3K8-siRNA and MAP3K8i regulate MAP3K8 blocking MAP3K8 signal luteinizing granulosa cells after estradiol promote function of progesterone synthesis is limited. These results indicated that MAP3K8 mediated regulation of estradiol on progesterone synthesis. Estradiol in most organizations through its nuclear receptor ERa and ER the beta function, but recent studies found that estradiol can also be its membrane receptor GPR30 binding function. In order to prove that estradiol modulates luteal progesterone synthesis Signal pathway and related molecular mechanism, we first analyzed the molecular estradiol receptor in luteal categories, the results show that ER alpha, ER beta and GPR30 were expressed in the corpus, and estradiol luteinizing granulosa cells in fast regulating MAP3K8 expression, and this effect may be membrane receptor inhibitor G15. It is proved that the expression of estradiol receptor GPR30 upregulated MAP3K8 function. At the same time, our experimental results show that MAP3K8 of luteal cells in regulating progesterone synthesis by promoting the phosphorylation of ERK. In conclusion, the expression of MAP3K8 in mouse ovarian corpus luteum, and the expression level in the mid luteal tissues were significantly higher in early stage and late luteal estradiol. The expression of MAP3K8 receptor GPR30 up-regulated in luteal cells, inhibition test showed that MAP3K8 mediated estradiol promote the physiological function of progesterone synthesis.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q492

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1 劉穎;蛋白激酶MAP3K8對(duì)小鼠黃體孕酮合成的調(diào)節(jié)機(jī)理研究[D];中國(guó)農(nóng)業(yè)大學(xué);2016年

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本文編號(hào)：1397099