叶酸缺乏对子宫内膜容受性相关基因表达的甲基化调控及其对胚胎着床的影响

发布时间：2018-05-07 07:06

本文选题：子宫内膜容受性 + 甲基化　；参考：《重庆医科大学》2012年博士论文

【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。
[Abstract]:Objective : To investigate whether folic acid is a group B vitamin which is transmitted by a carbon unit during the growth and development of mammals , which is a methyl donor with various methylation reactions .

Methods : The methylation patterns of Cdh1 , Pgr , Esr1 gene were analyzed by means of methylation specific PCR and sodium bisulfite sequencing . The expression of mRNA and protein of Cdh1 , Pgr , Esr1 gene was analyzed by means of real - time PCR , Western - blot and immunohistochemistry . The morphology and quantity of the cell - drinking process were detected by scanning electron microscopy .

Results : methylation analysis showed that the methylation rate of Esr1 gene promoter region decreased significantly in the absence of folic acid , but the methylation rate of Cdh1 and Pgr gene promoter did not change significantly . The expression of Cdh1 and Pgr gene was slightly higher than that of the normal control group , but the expression of Cdh1 and Pgr gene was slightly higher than that in the normal control group .

Conclusion : In this normal physiological process , the expression of DNA methylation may be regulated by DNA methylation , but the expression of Esr1 may be regulated by DNA methylation , but it does not affect the normal implantation of embryo .

Objective : To investigate whether folic acid plays an important role in the establishment and maintenance of placenta after implantation of embryo . It is very important for the establishment and maintenance of the placenta before the formation of placenta .

Methods : The embryo implantation model of pregnant rats and normal pregnant rats was established , and the birth of pregnant rats was analyzed by HE method . The expression of Hox10 and MMP2 in pregnant rats was analyzed by using HE method . The expression of apoptosis related factors Bax and Bcl 2 was analyzed by flow cytometry .

Results : The fetal birth rate of folic acid ( 0 , 0 / 20 ) was significantly lower than that of the control group ( 90 % , 18 / 20 ) .

Conclusion : In the absence of folic acid , the expression of Hox10 , MMP2 gene and the related genes Bax and Bcl 2 in decidualization of endometrium have been changed , and the expression of Bcl 2 and Bcl 2 in the endometrium have been altered . It is suggested that folic acid plays an important role in the process of decidualization of endometrium .

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R321

【参考文献】