小鼠月经模型中月经发生关键时期的确定及孕酮对相关基因表达的影响

发布时间：2019-01-04 20:46

【摘要】：在晚分泌期由于黄体功能的下降导致体内孕酮水平的降低称为孕酮撤退,孕酮撤退导致子宫内膜细胞经历一系列生化和细胞水平上的变化,最后发生凋亡和崩解,造成子宫内膜的出血和脱落,即所谓的月经发生。根据月经发生是否可被孕酮所逆转可将月经分为两个阶段：可被孕酮逆转的可逆阶段和和不可被孕酮逆转的不可逆阶段。前者为孕酮控制的信号通路,后者作为前者的后续阶段则触发了一系列组织崩解的作用因子。两阶段涉及完全不同的信号通路和级联反应体系。在人和灵长类解析上述分子机制有种种限制,因而如何获得适宜于进行活体研究的、廉价、易于操作的,特别是适合解析分子机理的动物模型是阐明月经发生机制的关键环节之一。因此确定小鼠月经模型中孕酮逆转的关键时期对于解析月经发生机制具有十分重要的意义。对此,我们进行了一些有益的探索,结果如下： 1.通过阴道细胞学监测出血和组织形态学鉴定,成功确定了小鼠生理性孕酮撤退月经模型中能被孕酮逆转的关键时期为孕酮撤退后的12-16h。 2.通过全基因组表达谱分析,比较小鼠生理性孕酮撤退模型中关键时期孕酮回植与孕酮撤退12h及16h的基因表达变化,找出差异表达基因,为后续研究提供导向。 3.在基因芯片数据的指导下,选择了MMP家族的主要成员、环氧合酶-2(COX-2)、VEGF作为研究的靶分子,从mRNA、蛋白水平及蛋白表达定位几个层面,探讨了在月经发生的关键时期内表达水平的变化,并比较了在关键时期前后回植孕酮对这些分子表达的影响。结果表明：在月经发生的早期阶段,孕酮回植可以降低MMP-3、MMP-10、MMP-11MMP-13及COX-2mRNA和蛋白表达。在月经发生的晚期,在关键时期前的12h回植孕酮,MMP-2、MMP-3、MMP-9、MMP-10、MMP-11、MMP-13和COX-2的mRNA和蛋白都受到强烈抑制；而关键时期后的16h回植孕酮则只能部分或不能减少这些MMP分子和COX-2的mRNA和蛋白的表达。 MMP-7的mRNA和蛋白表达不能被孕酮回植所抑制。提示MMP-7可能和小鼠子宫内膜的崩解无关。 VEGF mRNA和蛋白表达不受孕酮回植与否的影响,与其蛋白在小鼠子宫内膜的组织表达定位结果不符,这可能和VEGF表达的瞬时性增加有关。 4.探讨了孕酮回植对小鼠子宫内膜基质细胞凋亡的影响,发现在月经发生的关键时期之前的12h回植孕酮可以有效阻断子宫内膜细胞的凋亡,而关键时期之后的16h则不能起到阻断效应。同时分析了孕酮对凋亡相关基因p53、mdm2、bcl-2、bax、fas、caspase-3及caspase-8的mRNA和蛋白水平的表达的影响,初步证实孕酮回植的抗凋亡效应可能通过上调bcl-2/bax的比值从而抑制caspase-3表达有关。
[Abstract]:In the late secretory phase, progesterone level decreases due to the decrease of luteal function, which is called progesterone withdrawal. Progesterone withdrawal causes endometrial cells to undergo a series of biochemical and cellular changes, and finally apoptosis and disintegration occur. Causes endometrial bleeding and shedding, known as menstruation. According to whether menstruation can be reversed by progesterone, menstruation can be divided into two stages: reversible phase that can be reversed by progesterone and irreversible phase that can not be reversed by progesterone. The former is a progesterone-controlled signaling pathway, and the latter, as a follow-up stage, triggers a series of factors of tissue disintegration. The two stages involve completely different signal pathways and cascade reaction systems. In humans and primates, there are many limitations to understanding these molecular mechanisms, so how to get cheap, easy to operate, suitable for in vivo research, Especially the animal model which is suitable to explain the molecular mechanism is one of the key links to elucidate the mechanism of menstrual cycle. Therefore, it is very important to determine the key period of progesterone reversal in mouse menstrual model for elucidating the mechanism of menstruation. To this, we have carried on some beneficial exploration, the result is as follows: 1. By means of vaginal cytological monitoring and histomorphology identification, the key period of physiological progesterone withdrawal menstrual model in mice was determined to be 12-16 hours after progesterone withdrawal. 2. By analyzing the whole genome expression profile, we compared the gene expression changes between progesterone replantation and progesterone retreating 12h and 16h in the key period of mouse physiologic progesterone withdrawal model, and found the differentially expressed genes, which provided guidance for further study. 3. Under the guidance of gene chip data, the main member of the MMP family, cyclooxygenase-2 (COX-2), VEGF), was selected as the target molecule to locate the mRNA, protein level and protein expression. The changes of expression levels during the critical period of menstruation were studied, and the effects of progesterone reimplantation on the expression of these molecules were compared before and after the critical period. The results showed that progesterone reimplantation could decrease the expression of MMP-3,MMP-10,MMP-11MMP-13, COX-2mRNA and protein in the early stage of menstruation. At the late stage of menstruation, the reimplantation of progesterone, mRNA and protein of MMP-2,MMP-3,MMP-9,MMP-10,MMP-11,MMP-13 and COX-2 were strongly inhibited 12 hours before the critical period. However, the reimplantation of progesterone at 16 hours after the critical period could only partially or cannot reduce the expression of mRNA and protein of these MMP molecules and COX-2. The expression of mRNA and protein in MMP-7 could not be inhibited by progesterone replantation. The results suggest that MMP-7 may not be related to the disintegration of mouse endometrium. The expression of VEGF mRNA and protein was not affected by progesterone reimplantation or not, which was not consistent with the localization of VEGF mRNA protein in mouse endometrium, which might be related to the transient increase of VEGF expression. 4. The effect of progesterone reimplantation on apoptosis of mouse endometrial stromal cells was studied. It was found that the reimplantation of progesterone 12 hours before the critical period of menstruation could effectively block the apoptosis of endometrial cells. However, 16 hours after the critical period did not play a blocking effect. At the same time, the effect of progesterone on the expression of mRNA and protein of apoptosis-related gene p53 mdm2mdm2bcl-2baxanfascaspase-3 and caspase-8 was analyzed. It was preliminarily confirmed that the anti-apoptotic effect of progesterone replantation may inhibit the expression of caspase-3 by upregulating the ratio of bcl-2/bax.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R-332

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