叶酸缺乏对小鼠子宫内膜蜕膜化和胚胎发育的影响及其对基因组甲基化模式的调控

发布时间：2018-05-15 04:33

本文选题：叶酸 + 蜕膜化　；参考：《重庆医科大学》2016年博士论文

【摘要】：目的:妊娠结局与环境因素密切相关,母体饮食可通过表观遗传调控对胚胎发育及子代的表型产生影响。叶酸作为哺乳动物不可或缺的营养元素,参与DNA合成及甲基化反应等重要生理过程。叶酸缺乏或代谢障碍可改变基因组甲基化模式,影响基因的表达或功能,导致病理过程的发生。以往关于叶酸在妊娠中的作用研究多集中于叶酸代谢酶与胚胎发育的关系,但现有研究成果在深入阐释叶酸缺乏诱发胚胎发育异常的分子机制中仍显不足,关于叶酸在妊娠早期子宫内微环境中的作用报道甚少。课题组前期研究发现母体低叶酸水平并未对子宫内膜容受性造成不良影响,因此,本研究旨在探讨以下问题:1.叶酸缺乏对后续子宫内膜功能即子宫内膜基质细胞的蜕膜化和胚胎发育的影响;2.叶酸缺乏是否会通过改变相关基因的甲基化模式对子宫内膜蜕膜化和胚胎发育产生不利影响?3.平面细胞极性信号通路(planar cell polarity pathway,PCP)作为神经管发育过程的关键信号通路,母体低叶酸水平是否影响其核心基因的表达与功能?本文将分别从母体与胚胎两方面阐明叶酸缺乏对妊娠过程的影响及其对基因组甲基化模式的调控,以全面了解叶酸在妊娠过程中的作用,为调整育龄女性补充叶酸的时机、完善叶酸作用的分子机制提供实验依据。方法:1.动物模型建立及标本收集:采用无叶酸饲料饲养小鼠5周,对照组正常饮食。采用电化学发光法检测小鼠血清叶酸水平,确定叶酸缺乏小鼠模型是否建立成功。正常妊娠模型:将雌鼠与正常性成熟雄鼠合笼交配,以次日查见阴栓记为孕1天(D1)。正常组与叶酸缺乏组小鼠于正常妊娠6到13天(D6-D13)脱颈处死,收集子宫内膜及胚胎组织。人工诱导蜕膜化模型:将雌鼠与结扎输精管的雄鼠合笼交配,以次日查见阴栓记为假孕第1天(PD1),小鼠假孕4天(PD4)一侧宫角注射玉米油诱导蜕膜化,对侧宫角不注射作为对照,正常组与叶酸缺乏组小鼠于PD8处死,收集子宫组织。2.叶酸缺乏效应观察:1)子宫内膜蜕膜化检测:对小鼠蜕膜化时期(D6-D8)子宫外观进行观察,统计蜕膜鼓包数目及直径。借助人工诱导蜕膜化模型进行体内功能实验,通过观察子宫外观、称量子宫湿重、HE染色观察子宫切片形态及Real-time PCR检测蜕膜化标志分子Bmp2、dt PRP的m RNA水平来衡量人工诱导蜕膜化是否成功,统计两组小鼠诱导成功率。体外功能实验通过分离小鼠原代子宫内膜基质细胞,利用雌孕激素诱导蜕膜化实现,采用Real-time PCR检测Bmp2、dt PRP的m RNA表达,利用免疫荧光检测蜕膜化标志分子Desmin的蛋白表达。2)生殖能力及胚胎发育状态检测:对两组小鼠进行饲养繁殖,比较产仔能力。观察D9-D13小鼠子宫外观和胚胎形态,利用HE染色观察宫腔内胚胎发育状态,统计子宫湿重和胚胎吸收率。3.基因组甲基化模式检测:采用简化代表性亚硫酸氢盐测序法(Reduced Representation Bisulphite Sequencing,RRBS)对正常组和叶酸缺乏组小鼠D6-D8子宫内膜、D9-D11胚胎组织的全基因组甲基化模式进行检测,分析其甲基化差异,统计两组之间存在的差异甲基化区域(Differentially Methylated Regions,DMRs),利用生物信息学方法(GO分析)对差异甲基化基因进行功能聚类。采用Real-time PCR对测序结果中涉及的差异甲基化基因的m RNA水平进行检测。4.PCP信号通路分析:分别采用Real-time PCR、Western blot对两组小鼠D9-D12胚胎组织中PCP信号通路核心基因Vangl(Vangl1和Vangl2)的m RNA、蛋白水平进行检测。采用免疫共沉淀方法检测正常组和叶酸缺乏组小鼠D9、D10胚胎组织中Vangl基因与其配体Dvl蛋白的相互作用。采用亚硫酸氢盐测序(BSP)法对两组小鼠D9胚胎组织中Vangl1、Vangl2基因启动子区和第一外显子区的甲基化状态进行检测。利用直接测序法对两组小鼠D9-D13胚胎组织中Vangl基因(Vangl1和Vangl2)CDS区进行测序分析,再经比对NCBI公共数据库,筛选Vangl基因突变或SNP位点。结果:1.叶酸缺乏对子宫内膜蜕膜化的影响:蜕膜化时期子宫外观观察结果显示,与正常组相比,叶酸缺乏组小鼠蜕膜鼓包数目减少、直径较小。人工诱导蜕膜化模型统计结果显示,叶酸缺乏组小鼠成功率仅15%;诱导侧宫角湿重较正常组小鼠减轻;HE染色结果显示正常组小鼠诱导侧可见体积增大、双核或多核的蜕膜细胞,叶酸缺乏组小鼠无类似变化;Real-time PCR结果表明,叶酸缺乏组小鼠诱导蜕膜化后,蜕膜化marker Bmp2、dt PRP的m RNA水平较正常组小鼠显著降低。体外功能实验显示,正常组小鼠基质细胞经激素作用72h后,细胞变大,由长梭形变为多角形,而叶酸缺乏状态下,基质细胞仍呈成纤维细胞样;Real-time PCR结果显示,叶酸缺乏组小鼠基质细胞经激素诱导后Bmp2、dt PRP的m RNA较正常组细胞表达减少;免疫荧光结果显示,蜕膜化marker Desmin蛋白阳性信号在叶酸缺乏组细胞显著减弱。2.叶酸缺乏对胚胎发育的影响:饲养结果表明,叶酸缺乏状态下的小鼠产仔窝数及产仔个数显著减少。叶酸缺乏组小鼠自D10开始出现子宫出血,D11出血加重,D12、D13胚胎吸收明显。与正常组相比,叶酸缺乏组小鼠子宫湿重自D10开始下降,随妊娠进行下降更为明显。叶酸缺乏组小鼠的胚胎吸收率高达70%。正常组小鼠胚胎随妊娠天数增加生长分化呈现可辨认的仔鼠形态,而叶酸缺乏组小鼠胚胎出现生长粘连、分化发育异常。3.叶酸缺乏对基因组甲基化模式的影响:RRBS结果显示,两组小鼠子宫内膜基因组在D6、D7启动子区和CGI区的不同类型m C(包括m CG和m CHG,m CHH,其中H代表A、G或T)分布比例类似,D8叶酸缺乏组小鼠m CG比例增加而m CHH比例下降;胚胎组织基因组在D9、D10启动子区和Cp G岛(CGI)区的不同类型m C分布比例类似,D11叶酸缺乏组小鼠m CG比例增加而m CHH比例下降。与正常组相比,叶酸缺乏组小鼠子宫内膜组织中总C的平均甲基化水平在D7开始下降,D8下降更为明显,而m C的平均甲基化水平则在D8出现降低;胚胎组织基因组总C的平均甲基化水平在孕10天开始下降,孕11天下降更为明显,而m C的平均甲基化水平在D9-D11均高于正常组,但m CG在叶酸缺乏组的甲基化水平仍有所降低;甲基化水平的差异均集中于CG和m CG。对两组之间的差异甲基化区域统计结果显示,D6、D7和D8子宫内膜组织基因组分别存在666个、646个和785个DMRs;D9、D10和D11胚胎组织基因组分别存在795个、2480个和1602个DMRs。GO分析结果显示,D6-D8差异甲基化区域相关基因主要涉及生物粘附、生物调节、细胞增殖、发育代谢和信号通路功能;D9-D11差异甲基化区域相关基因主要涉及生物调节、细胞过程、发育代谢、应激反应和信号通路功能。Real-time PCR结果显示,两组间存在甲基化差异的基因,其m RNA水平也发生变化。4.叶酸缺乏对PCP信号通路的影响:Real-time PCR、Western blot结果显示,叶酸缺乏组小鼠胚胎组织中Vangl1和Vangl2的m RNA及蛋白水平自D9开始出现下降,并随妊娠进行下调更为明显。免疫共沉淀结果显示,叶酸缺乏的情况下,小鼠胚胎组织中Vangl1蛋白与Dvl1的相互作用被抑制,而与Dvl2、Dvl3的结合未受影响;而Vangl2蛋白与Dvl1、Dvl2、Dvl3的结合均被叶酸缺乏抑制。BSP测序结果显示,叶酸缺乏组小鼠D9胚胎组织中Vangl基因甲基化率较正常组无显著差异。CDS测序结果与NCBI数据库比对发现,正常组小鼠和叶酸缺乏组小鼠D9-D13胚胎组织中Vangl基因存在三个SNP位点,分别是Vangl1基因SNP位点rs36584696、Vangl2基因SNP位点rs48000091和rs31578570。结论:1.叶酸缺乏抑制小鼠子宫内膜基质细胞的蜕膜化进程,同时引起子宫内膜基因组甲基化模式的改变,包括不同类型m C的分布和平均甲基化水平,从而影响关键基因的表达和功能,这可能是低叶酸水平导致蜕膜化进程受阻的潜在分子机制。母体叶酸摄入不足在妊娠早期即发挥作用,以上数据为调整补充叶酸的时机提供了基础研究证据。2.叶酸缺乏会导致小鼠生殖能力严重受损,胚胎发育停滞,同时改变胚胎基因组的甲基化模式,包括不同类型m C的分布和甲基化水平,影响发育相关基因的表达。3.叶酸缺乏会抑制PCP信号通路核心基因Vangl基因的表达及功能,其作用机制不依赖于DNA甲基化,叶酸摄入不足可能不会引起Vangl基因发生突变。基因组甲基化模式的改变,导致发育关键基因的表达和功能异常以及PCP信号通路的抑制,这可能是叶酸缺乏损害胚胎发育的潜在分子机制。
[Abstract]:Objective: pregnancy outcome is closely related to environmental factors. Maternal diet can affect embryo development and progeny phenotype through epigenetic regulation. Folic acid, as an indispensable nutrient element in mammals, is involved in important physiological processes such as DNA synthesis and methylation. Folic acid deficiency or metabolic disorders can change the mode of genome methylation. The effect of the expression or function of the gene on the pathogenesis of the pathological process. The previous research on the role of folic acid in pregnancy is mainly focused on the relationship between folate metabolism enzyme and embryo development. However, the existing research results are still insufficient in explaining the molecular mechanism of folic acid deficiency inducing embryo development abnormality. There are few reports in the environment. Previous studies have found that maternal low folate levels do not affect endometrial receptivity. Therefore, this study aims to explore the following questions: 1. the effects of folic acid deficiency on subsequent endometrial function, decidua and embryonic development of endometrial stromal cells, 2. folic acid deficiency By changing the methylation patterns of related genes adversely affecting endometriosis and embryonic development? The 3. plane cell polarity signal pathway (planar cell polarity pathway, PCP) is the key signal pathway for the development of the neural tube. Does the maternal low folate level affect the expression and function of its core genes? This article will be different The effects of folic acid deficiency on the pregnancy process and the regulation of methylation patterns of genome are clarified from two aspects of mother and embryo, in order to fully understand the role of folic acid in pregnancy, to provide experimental basis for adjusting the timing of folic acid supplementation for women of childbearing age and to improve the molecular mechanism of folic acid action. Methods: the establishment of 1. animal models and collection of specimens: The mice were fed with folic acid for 5 weeks and the control group had a normal diet. The level of serum folic acid in mice was detected by electrochemiluminescence. The mice model of folic acid deficiency was established successfully. The normal pregnancy model: the female rats were copated with the normal mature male rats, and the second day was found to be 1 days of pregnancy (D1). The normal group and the folic acid deficiency group were small. Rats were removed from the normal pregnancy for 6 to 13 days (D6-D13), and the endometrium and embryonic tissue were collected. Artificial decidualic model was induced by artificial decidua model: the female rats were copated with the male rats with the vasectomy, and the second day was found to be first days (PD1), and the mice were injected with corn oil to induce decidua by injection of corn oil on one side of the mouse 4 days (PD4). In the normal group and the folic acid deficiency group, the mice were killed at PD8 and the.2. folate deficiency effect was observed. 1) endometrial decidualization detection: the appearance of the uterus in the period of decidualization (D6-D8) in mice was observed, the number and diameter of the decidua drum package were statistically analyzed. Weighing uterus wet weight, HE staining observation of uterine section morphology and Real-time PCR detection of deciduating marker molecule Bmp2, DT PRP m RNA level to measure the success of artificial deciduation, statistics two groups of mice induced success rate. In vitro functional experiment by separating the primary endometrial stromal cells in mice and using estrogen and progesterone to induce decidua. At present, Real-time PCR was used to detect Bmp2, m RNA expression of DT PRP, detection of the reproductive ability and embryonic development state of the decidua marker molecule Desmin by immunofluorescence. The two groups of mice were bred and bred to compare the offspring's ability. The appearance of uterus and the embryo of D9-D13 mice were observed and the uterine embryos were observed by HE staining. The.3. genomic methylation pattern of uterine wet weight and embryo absorption rate was detected by the simplified representative hydrogen sulphite sequencing method (Reduced Representation Bisulphite Sequencing, RRBS) for the detection of complete genomic methylation patterns in the normal and folic acid deficient mice D6-D8 endometrium and D9-D11 embryo fetal tissues. The difference of methylation was analyzed, and the difference methylation area (Differentially Methylated Regions, DMRs) existed between the two groups. The differential methylation gene was clustered by Bioinformatics Method (GO analysis). Real-time PCR was used to detect the m RNA level of the differential methylation genes involved in the sequencing results and the.4.PCP signal was detected. Path analysis: Real-time PCR and Western blot were used to detect the m RNA and protein level of the PCP signaling pathway core gene Vangl (Vangl1 and Vangl2) in the two groups of D9-D12 embryonic tissues, and the immunoprecipitation method was used to detect the normal group and the folate deficiency group mice D9. Using the method of hydrogen sulfite sequencing (BSP), the methylation status of the Vangl1, Vangl2 gene promoter and the first exon of the two groups of D9 embryos was detected. The CDS region of the Vangl gene (Vangl1 and Vangl2) in the D9-D13 embryo tissues of the two groups of mice was sequenced and analyzed by direct sequencing, and then compared to the NCBI public database. Vangl gene mutations or SNP loci were selected. Results: the effect of 1. folate deficiency on endometriosis: the appearance of uterine appearance during the deciduate period showed that the number of decidua drums in the folic acid deficiency group decreased and the diameter was smaller compared with the normal group. The results of the artificial decidua model showed that the rate of success in the folic acid deficiency group was only 15%. The results of HE staining showed that the volume of the induced side of the normal group was larger than that of the normal group, and there was no similar change in the decidua cells in the folic acid deficiency group. The results of Real-time PCR showed that the m RNA level of the deciduated marker Bmp2 and DT PRP in the folic acid deficiency group was more than that of the normal mice. In vitro functional experiments showed that after the hormone action of 72h in the normal group, the cells became larger and the spindle deformation was polygonal, and the matrix cells were fibroblast like in the absence of folic acid. The results of Real-time PCR showed that the mouse base cells in the folic acid deficiency group were induced by hormone Bmp2, and the m RNA of DT PRP was more than the normal group. The results of immunofluorescence showed that the deciduate marker Desmin positive signal significantly weakened the effect of folate deficiency on the development of.2. folate deficiency in folic acid deficiency group: the feeding results showed that the number of litter and the number of litter in the mice under the folic acid deficiency were significantly reduced. The mice in the folic acid deficiency group began to appear from the uterus from D10. Blood, D11 bleeding aggravated, D12, D13 embryo absorption obvious. Compared with the normal group, the wet weight of the uterus in the folic acid deficiency group began to decrease from the D10, and decreased with the pregnancy. The embryo absorption rate of the folic acid deficiency mice was higher than that of the normal group of 70%.. The effects of.3. folate deficiency on genomic methylation patterns were found in the mice in the spent group. The RRBS results showed that the endometrial genomes of the two groups of mice were in the different types of M C in D6, D7 promoter and CGI region (including m CG and m CHG. The proportion of M CHH decreased in proportion, and the proportion of M C distribution in D9, D10 promoter and Cp G Island (CGI) region was similar. The proportion of M CG in the mice with folic acid deficiency group increased and the proportion of M decreased. The 8 decrease was more obvious, while the average methylation level of M C decreased in D8; the average methylation level of total C in the embryo tissue began to decrease at the 10 day of pregnancy, and the 11 day of pregnancy decreased more obviously, while the average methylation level of M C was higher in D9-D11 than in the normal group, but the methylation level of M CG in the folic acid deficiency group was still lower. The difference in level was concentrated on the difference between CG and m CG. for the difference methylation between the two groups. The genomic DNA of the endometrium in the D6, D7 and D8 was 666, 646 and 785 DMRs, 795 in D9, D10 and D11 embryo, respectively. 2480 and 1602 DMRs.GO analysis showed that D6-D8 differential methylation regions were related. Genes mainly involve biological adhesion, biological regulation, cell proliferation, developmental metabolism and signaling pathway. D9-D11 differentially methylated region related genes mainly involve biological regulation, cell process, developmental metabolism, stress response and signaling pathway function.Real-time PCR results show that the two groups have different methylation genes, and their m RNA levels are also The effects of changes in.4. folate deficiency on PCP signaling pathways: Real-time PCR, Western blot results showed that m RNA and protein levels of Vangl1 and Vangl2 in the folic acid deficient group decreased from D9 to D9, and decreased with pregnancy. The immunoprecipitation results showed that the mouse embryo group was deficient in folic acid. The interaction between Vangl1 protein and Dvl1 was inhibited, and the combination of Dvl2 and Dvl3 was not affected, while the combination of Vangl2 protein and Dvl1, Dvl2, Dvl3 were all shown by the inhibition of folic acid deficiency and.BSP sequencing. There was no significant difference in the methylated rate between the Vangl gene in the folic acid deficiency mice and the normal group of the Vangl gene in the D9 embryo tissue of the mice. It was found that there were three SNP loci in the D9-D13 embryo tissue of normal mice and folic acid deficiency mice, which were Vangl1 SNP loci rs36584696, Vangl2 gene SNP loci rs48000091 and rs31578570. conclusion: 1. folate deficiency inhibited the decidua process of endometrial stromal cells in mice, and the endometrium genome was caused. Changes in methylation patterns, including the distribution and average methylation level of different types of M C, affect the expression and function of key genes, which may be a potential molecular mechanism for the inhibition of decidua process by low folate levels. Maternal folic acid intake plays a role in the early pregnancy, and the above data are the time to adjust the timing of folic acid supplementation. Basic research evidence,.2. folate deficiency, can cause severe damage to the reproductive capacity of mice, stagnation of embryo development, and change the methylation patterns of the embryo genome, including the distribution of different types of M C and the level of methylation, and the expression of.3. folate deficiency will inhibit the expression of the Vangl gene of the core gene of the PCP signaling pathway. And its function is not dependent on DNA methylation, and the deficiency of folic acid may not cause the mutation of the Vangl gene. The alteration of the methylation pattern of the genome leads to the expression and function abnormality of the key developmental genes and the inhibition of the PCP signaling pathway, which may be a potential molecular mechanism for the deficiency of folic acid to damage the development of embryos.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714

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