蛋白激酶MAP3K8对小鼠黄体孕酮合成的调节机理研究

发布时间：2018-01-08 12:21

本文关键词：蛋白激酶MAP3K8对小鼠黄体孕酮合成的调节机理研究　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

更多相关文章： MAP3K-8 雌二醇 雌二醇膜受体 黄体孕酮

【摘要】：黄体是卵泡排卵后形成的一个短暂性的分泌器官,在调节发情周期及妊娠维持过程中发挥重要的作用。黄体的主要功能是通过合成及分泌孕酮调节多种生理活动,而黄体中孕酮的合成受催乳素、促黄体素、雌二醇、前列腺素等激素的调节。在啮齿类动物中,催乳素和雌二醇是促进孕酮合成的主要激素,促黄体素在小鼠黄体中调节孕酮合成的作用较小。大量的研究证明了雌二醇在调节黄体孕酮合成的过程中起着非常关键的作用,但对其作用机理仍不清楚。本实验室之前的研究发现MAP3K8与激素的合成与分泌有关。因此,本研究的主要目标是研究MAP3K8在小鼠黄体中的表达,并进一步的探索该基因对于孕酮合成的调节作用及机理。首先利用免疫组织化学(IHC)方法检测了MAP3K8在小鼠卵巢中的表达,结果显示MAP3K8特异性的在卵巢黄体细胞表达,颗粒细胞及膜细胞中未检测到阳性信号。RT-qPCR及WB方法对不同时期的黄体中MAP3K8表达的分析结果证明MAP3K8在中期黄体中的表达量显著高于早期及晚期黄体。为了进一步探讨MAP3K8对黄体功能的调节,利用MAP3K8-siRNA及MAP3K8化学抑制剂(MAP3K8 inhibitor, MAP3K8i)阻断黄体化颗粒细胞中MAP3K8信号,结果发现黄体化颗粒细胞中孕酮的分泌水平降低。体内注射MAP3K8i的实验结果与细胞培养的结果一致,即注射MAP3K8i导致小鼠血清及黄体组织中孕酮的含量降低。之后的研究发现注射MAP3K8i后孕鼠黄体中孕酮水平的变化会进一步的影响胚胎着床。以上实验结果证明MAP3K8在调节黄体孕酮合成及相应的孕酮生理功能中有着重要的作用。此外,本研究发现,雌二醇能够上调黄体细胞中MAP3K8的表达,促黄体素及催乳素均不能调节MAP3K8的表达。MAP3K8-siRNA及MAP3K8i阻断黄体化颗粒细胞中MAP3K8信号后,雌二醇促进孕酮合成的功能受到了限制。这些实验结果说明MAP3K8介导雌二醇对孕酮合成的调节功能。多数组织中雌二醇通过其核受体ERa及ERβ发挥功能,但近期的研究发现雌二醇也可与其膜受体GPR30结合发挥功能。为了证明雌二醇调节黄体孕酮合成的信号通路和相关的分子机理,我们首先分析了黄体中雌二醇受体的分子类别,结果表明ERα、ERβ及GPR30均在黄体中表达,并且黄体化的颗粒细胞中雌二醇能够快速的调节MAP3K8的表达,且这种作用可被膜受体抑制剂G15阻断,由此证明了雌二醇通过膜受体GPR30上调MAP3K8的表达而发挥功能。同时我们的实验结果证明MAP3K8对黄体细胞中孕酮合成的调节是通过促进ERK磷酸化实现的。综上所述,MAP3K8在小鼠卵巢的黄体中表达,并且在中期黄体中的表达量显著高于早期及晚期黄体。雌二醇通过膜受体GPR30上调黄体细胞中MAP3K8的表达,抑制实验证明MAP3K8介导了雌二醇促进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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q492

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