本文選題：葉酸缺乏 + 抑制　； 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：眾所周知,孕婦葉酸缺乏會(huì)引起一系列不良的妊娠結(jié)局,包括胚胎畸形、流產(chǎn)、早產(chǎn)、低出生體重兒等,其中,葉酸缺乏與神經(jīng)管畸形的關(guān)系已被廣大學(xué)者所認(rèn)同。在已有的研究中,人們更多地關(guān)注葉酸缺乏對(duì)胚胎發(fā)育方面的影響,而少有人關(guān)注其對(duì)母體本身的不良影響。一次成功的妊娠除了需要正常發(fā)育的胎兒以外,孕母子宮內(nèi)膜容受性的建立以及基質(zhì)細(xì)胞正常發(fā)生蛻膜化也極其重要。課題組在前期研究中已證實(shí)葉酸缺乏對(duì)小鼠子宮內(nèi)膜容受性相關(guān)基因表達(dá)沒有影響,胚胎能夠正常著床,但最終妊娠結(jié)局不良,葉酸缺乏孕鼠出現(xiàn)胚胎丟失增多、胚胎體積減小的情況。因此,本文旨在研究葉酸缺乏對(duì)胚胎著床以后的重要分子事件—子宮內(nèi)膜蛻膜細(xì)胞凋亡的影響,并進(jìn)一步探討在葉酸缺乏孕鼠中子宮內(nèi)膜蛻膜化進(jìn)程是否受損,為全面深入認(rèn)識(shí)葉酸缺乏所致出生缺陷的機(jī)理提供更多的實(shí)驗(yàn)證據(jù)。方法：分別建立正常小鼠妊娠第7天、8天和葉酸缺乏小鼠妊娠第7天、8天模型,進(jìn)行以下實(shí)驗(yàn)：①采用流式細(xì)胞術(shù)、線粒體膜電位檢測(cè)試劑盒(JC-1)和TUNEL方法檢測(cè)孕鼠子宮內(nèi)膜蛻膜細(xì)胞凋亡；②透射電鏡觀察蛻膜細(xì)胞線粒體及內(nèi)質(zhì)網(wǎng)形態(tài);③免疫組化檢測(cè)Bax、 Bcl2的表達(dá)及分布；④ Western blot檢測(cè)凋亡相關(guān)基因Bax、Bcl2、cleaved-Caspase3、pro-Caspase3、cytochrome c的表達(dá)；⑤免疫熒光、激光共聚焦顯微鏡觀察細(xì)胞色素c的釋放；⑥ Real Time-PCR和Western blot檢測(cè)蛻膜化相關(guān)基因Hoxa10、BMP2、MMP2、MMP9的表達(dá)。結(jié)果：葉酸缺乏組孕鼠蛻膜細(xì)胞線粒體腫脹、內(nèi)質(zhì)網(wǎng)擴(kuò)張不及正常組明顯,流式細(xì)胞術(shù)、TUNEL、JC-1檢測(cè)均證實(shí)葉酸缺乏組蛻膜細(xì)胞凋亡降低,Western blot結(jié)果顯示葉缺組蛻膜組織Bax、 cleaved-Caspase3表達(dá)降低而pro-Caspase3、Bcl2在兩組的表達(dá)無明顯差異。Bax、Bcl2免疫組化結(jié)果與Western blot結(jié)果一致。cytochrome c免疫熒光、激光共聚焦顯微鏡觀察結(jié)果示葉酸缺乏組cytochrome c從線粒體內(nèi)釋放到細(xì)胞漿的量較正常組少。葉酸缺乏組蛻膜組織蛻膜化相關(guān)基因Hoxa10、BMP2、MMP2、MMP9表達(dá)降低。結(jié)論：在蛻膜化過程中,葉酸缺乏鼠蛻膜細(xì)胞凋亡受到抑制,蛻膜細(xì)胞凋亡減少。同時(shí),線粒體凋亡途徑中的相關(guān)基因表達(dá)在正常組和葉酸缺乏組之間有明顯差異。實(shí)驗(yàn)結(jié)果表明葉酸缺乏可能通過抑制線粒體凋亡途徑減少蛻膜細(xì)胞凋亡,進(jìn)而影響孕鼠子宮內(nèi)膜蛻膜化進(jìn)程。
[Abstract]:Objective: it is well known that maternal folic acid deficiency can lead to a series of adverse pregnancy outcomes, including embryo malformation, abortion, premature delivery, low birth weight infants, etc. Among them, the relationship between folic acid deficiency and neural tube malformation has been recognized by many scholars. In previous studies, more attention has been paid to the effects of folic acid deficiency on embryonic development, while less attention has been paid to the adverse effects of folic acid deficiency on the maternal body itself. The establishment of endometrial receptivity and the normal decidualization of stromal cells are also very important for a successful pregnancy. In previous studies, the research group has confirmed that folic acid deficiency has no effect on the expression of endometrial reception-related genes in mice, and the embryo can be implanted normally, but in the end the pregnancy outcome is poor, and the embryo loss increases in the pregnant mice with folic acid deficiency. A reduction in the size of an embryo. Therefore, the purpose of this study was to study the effect of folic acid deficiency on the apoptosis of decidua cells after embryo implantation, and to explore whether the decidualization process of endometrium was impaired in pregnant rats with folic acid deficiency. To provide more experimental evidence for understanding the mechanism of birth defects caused by folic acid deficiency. Methods: the models of normal mice on the 7th day of pregnancy and folic acid deficiency mice on the 7th day of pregnancy were established respectively. Flow cytometry was used in the following experiment: 1. Mitochondrial membrane potential detection kit (JC-1) and TUNEL method were used to detect apoptosis of decidua cells in pregnant rats. Transmission electron microscope was used to observe the expression and distribution of Baxand Bcl2 in decidual cells. (4) Western blot was used to detect the expression of apoptosis-related gene Baxia Bcl2cleaved-Caspase3 and pro-Caspase3 cytochrome c. The release of cytochrome c was observed by laser confocal microscopy and the expression of decidua-associated gene BMP2mMP2mMP9 was detected by Western blot. Results: mitochondria of decidua cells were swollen and endoplasmic reticulum dilatation was not obvious in the pregnant rats with folic acid deficiency group. The results of flow cytometry showed that the apoptosis of decidua cells decreased in folic acid deficiency group. The results of Western blot showed that the expression of Baxand cleaved-Caspase3 in decidua tissue was decreased, but the expression of pro-Caspase3 and Bcl2 in decidua was not significantly different between the two groups. The immunohistochemical results of Baxia Bcl2 were consistent with the results of Western blot. The results of laser confocal microscopy showed that the amount of cytochrome c released from mitochondria to cytoplasm in folic acid deficient group was less than that in normal group. The expression of decidualization related gene Hoxa10, BMP2, MMP2 and MMP9 was decreased in decidua deficient group. Conclusion: in the process of decidualization, the apoptosis of decidua cells was inhibited and the apoptosis of decidua cells was decreased in rats with folic acid deficiency. At the same time, the expression of related genes in mitochondrial apoptosis pathway was significantly different between normal group and folic acid deficiency group. The results showed that folic acid deficiency may reduce decidua cell apoptosis by inhibiting mitochondrial apoptosis and then affect the decidualization process in pregnant rats.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R714

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