【作者】 高茹菲；

【导师】 王应雄；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2012， 博士

【摘要】 目的：叶酸是哺乳类动物生长发育过程中担负一碳单位传递的B族维生素，是机体各种甲基化反应的甲基供体。叶酸摄入不足或叶酸代谢障碍时，可引起基因组DNA固有甲基化模式的改变而促进基因表达或抑制基因表达，诱发疾病状态，通过表观遗传修饰这一途径参与多种生命活动过程中的基因表达调控。但迄今为止，对于在妊娠早期子宫内膜容受性建立过程中，低叶酸水平是否会通过表观遗传修饰（DNA甲基化）来调控子宫内膜容受性相关基因的表达，进而影响胚胎着床，目前未见任何相关报道。因此，本研究旨在探索孕期低叶酸水平是否会影响子宫内膜容受性相关基因（Cdh1、Pgr、Esr1）DNA甲基化模式及其基因表达的改变，从而对胚胎着床产生影响。方法：建立叶酸缺乏孕鼠和正常孕鼠胚胎着床模型。采用甲基化特异性PCR和亚硫酸氢钠测序方法对Cdh1、Pgr、Esr1基因启动子区的甲基化模式进行分析。采用Real-time PCR、Western-blot、免疫组化对Cdh1、Pgr、Esr1基因的mRNA和蛋白表达进行分析。采用扫描电镜方法对子宫内膜容受性建立的形态学标志物—胞饮突的形态和数量进行检测。孕鼠尾静脉注射台盼蓝后计数孕鼠妊娠第5天的胚胎着床点数量。结果:甲基化分析结果显示叶酸缺乏情况下Esr1基因启动子区的甲基化率显著降低，而Cdh1、Pgr基因启动子的甲基化率没有发生显著改变。基因表达分析结果显示叶酸缺乏孕鼠组Esr1基因表达显著降低，而Cdh1、Pgr基因表达略高于正常对照组，但不具有统计学意义。扫描电镜结果显示叶酸缺乏情况下胞饮突的数量和形态均未发生明显改变。与正常对照组相比较，叶酸缺乏孕鼠组的妊娠率和妊娠第5天孕鼠的胚胎着床点数量均没有发生显著改变，胚胎正常着床。结论：在子宫内膜容受性建立这一正常生理过程中，DNA甲基化对容受性相关基因Cdh1、Pgr的表达调控作用并不明显，而Esr1的表达可能会受到DNA甲基化调控，但并不影响胚胎正常着床。叶酸缺乏下胚胎能够正常着床提示叶酸缺乏导致胚胎发育异常可能是着床以后的胚胎发育发生了异常。甲基化调控在肿瘤发生发展过程中与子宫内膜容受性建立这一正常生理过程中的不同作用，可能会为阐明肿瘤侵袭的“无控性”和胚胎着床的“有控性”机制提供新的线索。目的：胚胎着床以后子宫内膜蜕膜功能的正常表达是胎盘形成的重要基础，并在胎盘形成以前担负着保护和营养胚胎的重要作用，对妊娠的建立和维持至关重要。研究发现叶酸对胎盘的发育起着重要作用。但叶酸对着床后子宫内膜蜕膜化进程的影响以及叶酸缺乏是否会引起子宫内膜蜕膜化发生改变，从而造成胚胎发育异常，目前并不清楚，这值得我们去探索。因此，本研究旨在探索着床后子宫内膜蜕膜化是否会受到孕期低叶酸水平的影响。方法：建立叶酸缺乏孕鼠和正常孕鼠胚胎着床模型，对孕鼠胎儿的出生情况进行统计。采用HE方法检测孕鼠妊娠d7子宫内膜形态。采用Real-time PCR、Western-blot、免疫组化对孕鼠妊娠d7、d8子宫内膜Hoxa10、MMP2基因表达进行分析。采用流式细胞术检测妊娠d7子宫内膜的凋亡情况，并采用Western-blot对凋亡相关因子Bax、Bcl2的表达进行了分析。结果:叶酸缺乏孕鼠组胎儿出生率为（0，0/20）显著低于正常对照组（90%，18/20）。观察妊娠d7孕鼠子宫，叶酸缺乏孕鼠组（50%，4/8）胚胎呈现不同程度的吸收和发育阻滞现象，明显高于正常对照组（12.5%，1/8）。HE染色观察发现妊娠d7叶酸缺乏孕鼠组子宫内膜蜕膜形态发生改变。进一步检测发现叶酸缺乏情况下，在蜕膜化过程中起着重要作用的Hoxa10和MMP2基因表达显著降低。凋亡的正常维持对蜕膜化具有重要意义。检测发现叶酸缺乏孕鼠组妊娠d7子宫内膜的凋亡率明显低于正常对照组，且叶酸缺乏孕鼠组促凋亡因子Bax蛋白表达明显低于正常组，而抑凋亡因子Bcl2的表达却显著高于正常组。结论：研究发现叶酸缺乏情况下，在子宫内膜的蜕膜化中起着重要作用的Hoxa10、MMP2基因以及维持蜕膜化凋亡平衡的相关基因Bax、Bcl2表达均发生改变，子宫内膜蜕膜化受到损害，提示叶酸在子宫内膜蜕膜化过程中发挥着重要作用。更多还原

【Abstract】 Background: Folate, one of the B vitamins, provides the one-carbonunits required for methylation. Folate deficiency has been reported for theassociation with many pathologies. However, much less is known about theeffect of it on human reproduction, especially on implantation.Establishment of uterine receptivity is crucial for successful embryoimplantation. Gene expression can be influenced by both heredity andepigenetics such as DNA methylation. However, it is not known whether themethylation and expression of genes related to uterine receptivity can beaffected by folate levels. To explore whether folate deficiency affected theepigenetic regulation of genes related to uterine receptivity, and theirinfluence on implantation, we investigated the methylation and expressionof Cdh1, Pgr and Esr1during implantation and the implantation efficiency using a folate-deficient pregnant mouse model.Methods: A folate-deficient pregnant mouse model was established inthis study. Plasma folate levels of pregnant mice were detected using theelectro-chemiluminescence immunoassay. The methylation status of Cdh1,Pgr and Esr1promoter region was determined by methylation specific PCRand bisulfite sequencing. The expression of Cdh1, Pgr and Esr1in theimplantation-site endometrium was examined by real-time PCR, westernblot and immunohistochemistry. The number and the morphology ofpinopodes, important morphologic marker of endometrial receptivity, wereexamined using scanning-electron microscopy. The number of implantationsites on day5of pregnancy demarcated by distinct blue bands was recordedalso.Results: Here it showed that methylation status of Esr1wassignificantly decreased, while the methylation status of Cdh1and Pgr werenot varied in folate-deficient mice compared with wild type. The geneexpression of Esr1were significantly decreased, and Cdh1and Pgrexpression levels were slightly elevated in folate-deficient mice. Neither thenumber nor morphology of pinopodes was affected by folate deficiency.Furthermore, folate deficiency did not affect the number of implantationsites in mice.Conclusions: This study demonstrates for the first time that althoughthe methylation and expression of Esr1is significantly altered at the folate-deficient status, folate deficiency is not able to influence themethylation and expression of Pgr and Cdh1, two genes shown to beessential for uterine receptivity, and embryo can implant into endometriumnormally. The differential regulation of Esr1, Cdh1and Pgr methylationduring tumorigenesis and endometrium receptivity may be a new aspect forstudying the mechanisms of “uncontrolled” tumor invasion and “controlled”embryo implantation. It is a clue for further possibility of the effect of folateon pregnancy process after implantation, although embryo implantation isnormal at the deficiency of folate. Background: As is well known, uterine decidualization andplacentation is the basement of embryo development after embryoimplantation. It is believed that decidualization is much important for theprovision of nutrition to the developing embryo until the placenta has beendeveloped. The abnormal decidualization could not lead fetal get propernourishmen and develop normally. Studies have suggested that folate has aimportant role in the placentation. However, much less is known about theeffect of it on uterine decidualization. In this research, our aim is to study onthe possible roles of folate deficiency in the regulation of decidualization bycells apoptosis after embryo implantation.Methods: A folate-deficient pregnant mouse model was established inthis study and the fetus birth rate was analyzed. The morphologicalcharacteristics of uterine on day7was detected by HE staining. Theexpression of Hoxa10and MMP2in endometrium on day7and day8were examined by real-time PCR, western-blot and immunohistochemistry. Themouse uterine stromal cells on day7were isolated and purified forapoptosis by flow cytometry and the protein expression of Bax and Bcl2were examined using western-blot.Results: By mating normal or folate deficient females with normalmales, we found that the fetus birth rate (0,0/20) of folate deficient groupwas significantly decreased than that (90%,18/20) in control. The embryosin folate deficiency on day7were in different degrees of absorption. Theresults by HE staining showed the decidual morphology was affected byfolate deficiency. In folate-deficient mice, the gene expression of Hoxa10and MMP2were significantly decreased. Apoptosis of decidual tissues infolate deficiency was obviously decreased, and the Bax protein expressionwas significantly decreased while Bcl2was significantly elevated.Conclusions: This study demonstrates for the first time that expressionof Hoxa10and MMP2, two genes shown to be important fordecidualization, are significantly altered at the folate-deficient status, andthe expression of Bax and Bcl2, which play essential roles for apoptosis indecidualization, are also significantly altered in folate deficiency. Itsuggestes that folate may be essential for decidualization.更多还原