叶酸缺乏通过线粒体途径抑制孕鼠子宫内膜蜕膜细胞凋亡

发布时间：2018-05-24 19:04

本文选题：叶酸缺乏 + 抑制　；参考：《重庆医科大学》2015年硕士论文

【摘要】：目的：众所周知,孕妇叶酸缺乏会引起一系列不良的妊娠结局,包括胚胎畸形、流产、早产、低出生体重儿等,其中,叶酸缺乏与神经管畸形的关系已被广大学者所认同。在已有的研究中,人们更多地关注叶酸缺乏对胚胎发育方面的影响,而少有人关注其对母体本身的不良影响。一次成功的妊娠除了需要正常发育的胎儿以外,孕母子宫内膜容受性的建立以及基质细胞正常发生蜕膜化也极其重要。课题组在前期研究中已证实叶酸缺乏对小鼠子宫内膜容受性相关基因表达没有影响,胚胎能够正常着床,但最终妊娠结局不良,叶酸缺乏孕鼠出现胚胎丢失增多、胚胎体积减小的情况。因此,本文旨在研究叶酸缺乏对胚胎着床以后的重要分子事件—子宫内膜蜕膜细胞凋亡的影响,并进一步探讨在叶酸缺乏孕鼠中子宫内膜蜕膜化进程是否受损,为全面深入认识叶酸缺乏所致出生缺陷的机理提供更多的实验证据。方法：分别建立正常小鼠妊娠第7天、8天和叶酸缺乏小鼠妊娠第7天、8天模型,进行以下实验：①采用流式细胞术、线粒体膜电位检测试剂盒(JC-1)和TUNEL方法检测孕鼠子宫内膜蜕膜细胞凋亡；②透射电镜观察蜕膜细胞线粒体及内质网形态;③免疫组化检测Bax、 Bcl2的表达及分布；④ Western blot检测凋亡相关基因Bax、Bcl2、cleaved-Caspase3、pro-Caspase3、cytochrome c的表达；⑤免疫荧光、激光共聚焦显微镜观察细胞色素c的释放；⑥ Real Time-PCR和Western blot检测蜕膜化相关基因Hoxa10、BMP2、MMP2、MMP9的表达。结果：叶酸缺乏组孕鼠蜕膜细胞线粒体肿胀、内质网扩张不及正常组明显,流式细胞术、TUNEL、JC-1检测均证实叶酸缺乏组蜕膜细胞凋亡降低,Western blot结果显示叶缺组蜕膜组织Bax、 cleaved-Caspase3表达降低而pro-Caspase3、Bcl2在两组的表达无明显差异。Bax、Bcl2免疫组化结果与Western blot结果一致。cytochrome c免疫荧光、激光共聚焦显微镜观察结果示叶酸缺乏组cytochrome c从线粒体内释放到细胞浆的量较正常组少。叶酸缺乏组蜕膜组织蜕膜化相关基因Hoxa10、BMP2、MMP2、MMP9表达降低。结论：在蜕膜化过程中,叶酸缺乏鼠蜕膜细胞凋亡受到抑制,蜕膜细胞凋亡减少。同时,线粒体凋亡途径中的相关基因表达在正常组和叶酸缺乏组之间有明显差异。实验结果表明叶酸缺乏可能通过抑制线粒体凋亡途径减少蜕膜细胞凋亡,进而影响孕鼠子宫内膜蜕膜化进程。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R714

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