本文選題：葉酸 + 蛻膜化��； 參考：《重慶醫(yī)科大學》2016年博士論文

【摘要】：目的:妊娠結(jié)局與環(huán)境因素密切相關,母體飲食可通過表觀遺傳調(diào)控對胚胎發(fā)育及子代的表型產(chǎn)生影響。葉酸作為哺乳動物不可或缺的營養(yǎng)元素,參與DNA合成及甲基化反應等重要生理過程。葉酸缺乏或代謝障礙可改變基因組甲基化模式,影響基因的表達或功能,導致病理過程的發(fā)生。以往關于葉酸在妊娠中的作用研究多集中于葉酸代謝酶與胚胎發(fā)育的關系,但現(xiàn)有研究成果在深入闡釋葉酸缺乏誘發(fā)胚胎發(fā)育異常的分子機制中仍顯不足,關于葉酸在妊娠早期子宮內(nèi)微環(huán)境中的作用報道甚少。課題組前期研究發(fā)現(xiàn)母體低葉酸水平并未對子宮內(nèi)膜容受性造成不良影響,因此,本研究旨在探討以下問題:1.葉酸缺乏對后續(xù)子宮內(nèi)膜功能即子宮內(nèi)膜基質(zhì)細胞的蛻膜化和胚胎發(fā)育的影響;2.葉酸缺乏是否會通過改變相關基因的甲基化模式對子宮內(nèi)膜蛻膜化和胚胎發(fā)育產(chǎn)生不利影響?3.平面細胞極性信號通路(planar cell polarity pathway,PCP)作為神經(jīng)管發(fā)育過程的關鍵信號通路,母體低葉酸水平是否影響其核心基因的表達與功能?本文將分別從母體與胚胎兩方面闡明葉酸缺乏對妊娠過程的影響及其對基因組甲基化模式的調(diào)控,以全面了解葉酸在妊娠過程中的作用,為調(diào)整育齡女性補充葉酸的時機、完善葉酸作用的分子機制提供實驗依據(jù)。方法:1.動物模型建立及標本收集:采用無葉酸飼料飼養(yǎng)小鼠5周,對照組正常飲食。采用電化學發(fā)光法檢測小鼠血清葉酸水平,確定葉酸缺乏小鼠模型是否建立成功。正常妊娠模型:將雌鼠與正常性成熟雄鼠合籠交配,以次日查見陰栓記為孕1天(D1)。正常組與葉酸缺乏組小鼠于正常妊娠6到13天(D6-D13)脫頸處死,收集子宮內(nèi)膜及胚胎組織。人工誘導蛻膜化模型:將雌鼠與結(jié)扎輸精管的雄鼠合籠交配,以次日查見陰栓記為假孕第1天(PD1),小鼠假孕4天(PD4)一側(cè)宮角注射玉米油誘導蛻膜化,對側(cè)宮角不注射作為對照,正常組與葉酸缺乏組小鼠于PD8處死,收集子宮組織。2.葉酸缺乏效應觀察:1)子宮內(nèi)膜蛻膜化檢測:對小鼠蛻膜化時期(D6-D8)子宮外觀進行觀察,統(tǒng)計蛻膜鼓包數(shù)目及直徑。借助人工誘導蛻膜化模型進行體內(nèi)功能實驗,通過觀察子宮外觀、稱量子宮濕重、HE染色觀察子宮切片形態(tài)及Real-time PCR檢測蛻膜化標志分子Bmp2、dt PRP的m RNA水平來衡量人工誘導蛻膜化是否成功,統(tǒng)計兩組小鼠誘導成功率。體外功能實驗通過分離小鼠原代子宮內(nèi)膜基質(zhì)細胞,利用雌孕激素誘導蛻膜化實現(xiàn),采用Real-time PCR檢測Bmp2、dt PRP的m RNA表達,利用免疫熒光檢測蛻膜化標志分子Desmin的蛋白表達。2)生殖能力及胚胎發(fā)育狀態(tài)檢測:對兩組小鼠進行飼養(yǎng)繁殖,比較產(chǎn)仔能力。觀察D9-D13小鼠子宮外觀和胚胎形態(tài),利用HE染色觀察宮腔內(nèi)胚胎發(fā)育狀態(tài),統(tǒng)計子宮濕重和胚胎吸收率。3.基因組甲基化模式檢測:采用簡化代表性亞硫酸氫鹽測序法(Reduced Representation Bisulphite Sequencing,RRBS)對正常組和葉酸缺乏組小鼠D6-D8子宮內(nèi)膜、D9-D11胚胎組織的全基因組甲基化模式進行檢測,分析其甲基化差異,統(tǒng)計兩組之間存在的差異甲基化區(qū)域(Differentially Methylated Regions,DMRs),利用生物信息學方法(GO分析)對差異甲基化基因進行功能聚類。采用Real-time PCR對測序結(jié)果中涉及的差異甲基化基因的m RNA水平進行檢測。4.PCP信號通路分析:分別采用Real-time PCR、Western blot對兩組小鼠D9-D12胚胎組織中PCP信號通路核心基因Vangl(Vangl1和Vangl2)的m RNA、蛋白水平進行檢測。采用免疫共沉淀方法檢測正常組和葉酸缺乏組小鼠D9、D10胚胎組織中Vangl基因與其配體Dvl蛋白的相互作用。采用亞硫酸氫鹽測序(BSP)法對兩組小鼠D9胚胎組織中Vangl1、Vangl2基因啟動子區(qū)和第一外顯子區(qū)的甲基化狀態(tài)進行檢測。利用直接測序法對兩組小鼠D9-D13胚胎組織中Vangl基因(Vangl1和Vangl2)CDS區(qū)進行測序分析,再經(jīng)比對NCBI公共數(shù)據(jù)庫,篩選Vangl基因突變或SNP位點。結(jié)果:1.葉酸缺乏對子宮內(nèi)膜蛻膜化的影響:蛻膜化時期子宮外觀觀察結(jié)果顯示,與正常組相比,葉酸缺乏組小鼠蛻膜鼓包數(shù)目減少、直徑較小。人工誘導蛻膜化模型統(tǒng)計結(jié)果顯示,葉酸缺乏組小鼠成功率僅15%;誘導側(cè)宮角濕重較正常組小鼠減輕;HE染色結(jié)果顯示正常組小鼠誘導側(cè)可見體積增大、雙核或多核的蛻膜細胞,葉酸缺乏組小鼠無類似變化;Real-time PCR結(jié)果表明,葉酸缺乏組小鼠誘導蛻膜化后,蛻膜化marker Bmp2、dt PRP的m RNA水平較正常組小鼠顯著降低。體外功能實驗顯示,正常組小鼠基質(zhì)細胞經(jīng)激素作用72h后,細胞變大,由長梭形變?yōu)槎嘟切?而葉酸缺乏狀態(tài)下,基質(zhì)細胞仍呈成纖維細胞樣;Real-time PCR結(jié)果顯示,葉酸缺乏組小鼠基質(zhì)細胞經(jīng)激素誘導后Bmp2、dt PRP的m RNA較正常組細胞表達減少;免疫熒光結(jié)果顯示,蛻膜化marker Desmin蛋白陽性信號在葉酸缺乏組細胞顯著減弱。2.葉酸缺乏對胚胎發(fā)育的影響:飼養(yǎng)結(jié)果表明,葉酸缺乏狀態(tài)下的小鼠產(chǎn)仔窩數(shù)及產(chǎn)仔個數(shù)顯著減少。葉酸缺乏組小鼠自D10開始出現(xiàn)子宮出血,D11出血加重,D12、D13胚胎吸收明顯。與正常組相比,葉酸缺乏組小鼠子宮濕重自D10開始下降,隨妊娠進行下降更為明顯。葉酸缺乏組小鼠的胚胎吸收率高達70%。正常組小鼠胚胎隨妊娠天數(shù)增加生長分化呈現(xiàn)可辨認的仔鼠形態(tài),而葉酸缺乏組小鼠胚胎出現(xiàn)生長粘連、分化發(fā)育異常。3.葉酸缺乏對基因組甲基化模式的影響:RRBS結(jié)果顯示,兩組小鼠子宮內(nèi)膜基因組在D6、D7啟動子區(qū)和CGI區(qū)的不同類型m C(包括m CG和m CHG,m CHH,其中H代表A、G或T)分布比例類似,D8葉酸缺乏組小鼠m CG比例增加而m CHH比例下降;胚胎組織基因組在D9、D10啟動子區(qū)和Cp G島(CGI)區(qū)的不同類型m C分布比例類似,D11葉酸缺乏組小鼠m CG比例增加而m CHH比例下降。與正常組相比,葉酸缺乏組小鼠子宮內(nèi)膜組織中總C的平均甲基化水平在D7開始下降,D8下降更為明顯,而m C的平均甲基化水平則在D8出現(xiàn)降低;胚胎組織基因組總C的平均甲基化水平在孕10天開始下降,孕11天下降更為明顯,而m C的平均甲基化水平在D9-D11均高于正常組,但m CG在葉酸缺乏組的甲基化水平仍有所降低;甲基化水平的差異均集中于CG和m CG。對兩組之間的差異甲基化區(qū)域統(tǒng)計結(jié)果顯示,D6、D7和D8子宮內(nèi)膜組織基因組分別存在666個、646個和785個DMRs;D9、D10和D11胚胎組織基因組分別存在795個、2480個和1602個DMRs。GO分析結(jié)果顯示,D6-D8差異甲基化區(qū)域相關基因主要涉及生物粘附、生物調(diào)節(jié)、細胞增殖、發(fā)育代謝和信號通路功能;D9-D11差異甲基化區(qū)域相關基因主要涉及生物調(diào)節(jié)、細胞過程、發(fā)育代謝、應激反應和信號通路功能。Real-time PCR結(jié)果顯示,兩組間存在甲基化差異的基因,其m RNA水平也發(fā)生變化。4.葉酸缺乏對PCP信號通路的影響:Real-time PCR、Western blot結(jié)果顯示,葉酸缺乏組小鼠胚胎組織中Vangl1和Vangl2的m RNA及蛋白水平自D9開始出現(xiàn)下降,并隨妊娠進行下調(diào)更為明顯。免疫共沉淀結(jié)果顯示,葉酸缺乏的情況下,小鼠胚胎組織中Vangl1蛋白與Dvl1的相互作用被抑制,而與Dvl2、Dvl3的結(jié)合未受影響;而Vangl2蛋白與Dvl1、Dvl2、Dvl3的結(jié)合均被葉酸缺乏抑制。BSP測序結(jié)果顯示,葉酸缺乏組小鼠D9胚胎組織中Vangl基因甲基化率較正常組無顯著差異。CDS測序結(jié)果與NCBI數(shù)據(jù)庫比對發(fā)現(xiàn),正常組小鼠和葉酸缺乏組小鼠D9-D13胚胎組織中Vangl基因存在三個SNP位點,分別是Vangl1基因SNP位點rs36584696、Vangl2基因SNP位點rs48000091和rs31578570。結(jié)論:1.葉酸缺乏抑制小鼠子宮內(nèi)膜基質(zhì)細胞的蛻膜化進程,同時引起子宮內(nèi)膜基因組甲基化模式的改變,包括不同類型m C的分布和平均甲基化水平,從而影響關鍵基因的表達和功能,這可能是低葉酸水平導致蛻膜化進程受阻的潛在分子機制。母體葉酸攝入不足在妊娠早期即發(fā)揮作用,以上數(shù)據(jù)為調(diào)整補充葉酸的時機提供了基礎研究證據(jù)。2.葉酸缺乏會導致小鼠生殖能力嚴重受損,胚胎發(fā)育停滯,同時改變胚胎基因組的甲基化模式,包括不同類型m C的分布和甲基化水平,影響發(fā)育相關基因的表達。3.葉酸缺乏會抑制PCP信號通路核心基因Vangl基因的表達及功能,其作用機制不依賴于DNA甲基化,葉酸攝入不足可能不會引起Vangl基因發(fā)生突變�；蚪M甲基化模式的改變,導致發(fā)育關鍵基因的表達和功能異常以及PCP信號通路的抑制,這可能是葉酸缺乏損害胚胎發(fā)育的潛在分子機制。
[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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R714

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