叶酸缺乏孕鼠子宫内膜基因组甲基化模式改变及对基质细胞蜕膜化的抑制作用研究

Changes of genomic methylation patterns in endometrium of pregnant rats with folic acid deficiency and their inhibitory effects on decidualization of stromal cells

目的研究叶酸缺乏孕鼠子宫内膜基因组甲基化模式改变及对基质细胞蜕膜化的抑制作用。方法取30只性成熟SPF级小鼠,随机分为两组,实验组(15只)小鼠采用无叶酸饲料喂养,对照组(15只)小鼠给予正常饮食,均喂养4周。通过电化学发光法对小鼠血清叶酸含量进行检测,建立小鼠正常妊娠模型和人工诱导蜕膜化模型,检测子宫内膜蜕膜化的情况。通过免疫荧光法对蜕膜化分子标志物Desmin蛋白的表达水平进行检测,并用实时荧光定量PCR法对小鼠dt PRP-mRNA、Bmp2-mRNA的相对表达量进行检测。通过简化代表性亚硫酸氢盐测序法检测小鼠基因组甲基化模式。结果实验组小鼠血浆叶酸水平较对照组的水平明显下降(P<0.01)。实验组小鼠蜕膜鼓包数目少于对照组,直径小于对照组(P<0.05)。实验组小鼠人工诱导蜕膜化造模成功率仅为33.33%(3/15),而对照组人工诱导蜕膜化成功率为100.00%(15/15);诱导一侧宫角湿重轻于对照组(P<0.01);经HE染色结果可见实验组小鼠无明显变化,而对照组小鼠诱导侧体积增大,且出现双核或多核的蜕膜细胞。实时荧光定量PCR法检测结果发现,实验组小鼠经人工诱导蜕膜化后,其蜕膜化分子标志物dt PRP-mRNA、Bmp2-mRNA的相对表达量明显低于对照组(P<0.01)。体外功能实验结果发现,经激素作用3 d后,实验组小鼠基质细胞仍呈现成纤维细胞样,而对照组小鼠基质细胞形态改变,即由长梭形变为多角形,细胞增大明显。实时荧光定量PCR法检测结果发现,经激素诱导后,实验组小鼠基质细胞中dt PRP-mRNA、Bmp2-mRNA的相对表达量明显低于对照组(P<0.01)。经简化代表性亚硫酸氢盐测序法检测结果发现,甲基化胞嘧啶mC多数处在启动子区或CpG岛(CGI)中的CG位点,部分mC处在非CG位点,即mCHG与m CHH(H代表T、C或A)。两组小鼠在妊娠第6~7天时不同类型m C分布比较并无显著差异;妊娠第8天时,实验组小鼠mCG在启动子区与CGI中的比率明显高于对照组,而mCHH在启动子区与CGI中的比率低于对照组。结论叶酸缺乏不仅可阻滞小鼠子宫内膜基质细胞的蜕膜化进程,而且可改变子宫内膜基因组甲基化模式,如各类型的mC分布与平均甲基化水平等,进一步对关键基因的表达及功能造成影响,提示叶酸缺乏可损害小鼠孕早期子宫内膜功能,这可能是叶酸缺乏抑制蜕膜化进程的潜在分子机制。

Objective To study the changes of genomic methylation patterns in endometrium of pregnant rats with folic acid deficiency and its inhibitory effect on decidualization of stromal cells.Methods Thirty sexually mature SPF mice were randomly divided into experimental group(15 mice)fed with folic acid-free diet and control group(15 mice)fed with normal diet for 4 weeks.The serum folic acid content in mice was detected by electrochemiluminescence.The normal pregnancy model and artificial induced decidualization model were established to detect endometrial decidualization.The expression level of Desmin protein,a molecule marker of decidualization,was detected by immunofluorescence,and the relative expression levels of dtPRP-mRNA and Bmp2-mRNA in mice were detected by real-time fluorescence quantitative PCR.A simplified representative bisulfite sequencing method was used to detect genomic methylation patterns in mice.Results The plasma folic acid level in the experimental group was significantly lower than that in the control group(P<0.01).The number of decidual tympanic sacs in the experimental group was less than that in the control group,and the diameter was smaller than that in the control group(P<0.05).The success rate of artificial induced decidualization was only 33.33%(3/15)in the experimental group,and 100.00%(15/15)in the control group.The wet weight of the induced corners of the uterus was lighter than that of the control group(P<0.01).HE staining showed that there was no significant change in the experimental group,but the volume of the induced side increased in the control group,and there were binuclear or multinucleated decidual cells.The results of real-time fluorescence quantitative PCR showed that the relative expression of dtPRP-mRNA and Bmp2-mRNA in the experimental group was significantly lower than that in the control group(P<0.01).In vitro function test showed that after 3 days of hormone treatment,the stromal cells in the experimental group still showed fibroblast-like morphology,while in the control group,the stromal cells changed from long spindle to polygon,and the cells increased significantly.The results of real-time fluorescence quantitative PCR showed that after hormone induction,the relative expression of dtPRP-mRNA and Bmp2-mRNA in mouse stromal cells of the experimental group was significantly lower than that of the control group(P<0.01).The results of simplified representative bisulfite sequencing showed that most of methylated cytosine mCs were located at CG sites in promoter region or CpG island(CGI),and some mCs were located at non-CG sites,i.e.mCHG and mCHH(H stands for T,C or A).There was no significant difference in the distribution of different types of mC between the two groups at the 6th and 7th day of gestation.On the 8th day of gestation,the proportion of mCG in the promoter region and CGI of the experimental group was significantly higher than that of the control group,while the proportion of mCHH in the promoter region and CGI of the experimental group was lower than that of the control group.Conclusion Folic acid deficiency can not only block the decidualization process of mouse endometrial stromal cells,but also alter the genomic methylation pattern of endometrium,such as the distribution of mC and the average methylation level,which further affects the expression and function of key genes,suggesting that folic acid deficiency can impair the endometrial function of early pregnancy in mice,which may be a potential molecular mechanism for folic acid deficiency to inhibit decidualization.