孕期叶酸调节CDK5与TLR4甲基化改善幼年子代大鼠神经行为发育的研究

发布时间：2018-05-08 05:38

本文选题：叶酸 + DNA甲基化　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的孕期母体的营养状况与子代发育之间存在关联,叶酸作为一碳单位载体可参与体内多种甲基化反应,而DNA甲基化在子代个体神经系统发育中又起到重要作用。但孕期叶酸干预是否通过DNA甲基化途经调节子代神经发育,及其作用的具体基因位点目前尚不明确,本研究拟通过大鼠生殖实验,探讨孕期叶酸营养水平是否通过调节细胞周期蛋白依赖性激酶5(cyclin-dependent kinase 5,CDK5)、Toll样受体4(Toll-like receptors 4,TLR4)甲基化改善子代幼年大鼠神经发育,为孕期叶酸合理补充提供科学依据。方法40只SPF级雌性Sprague-Dawley(SD)大鼠随机分为4组:(1)叶酸缺乏组(folate-deficient diet group,DD)饲以叶酸缺乏饲料;(2)叶酸正常组(folate-normal diet group,ND)饲以叶酸正常饲料;(3)叶酸短期补充组(folate-added diet short-period group,FD-S)在合笼交配期饲以叶酸补充饲料10天,其余时间段饲以叶酸正常饲料;(4)孕前孕后叶酸全程补充组(folate-added diet long-period group,FD-L)全程饲以叶酸补充饲料。孕鼠分娩当日记作子代新生鼠出生后第1天(Postnatal day 1,PD1),于PD1-PD14,每日监测仔鼠的体重变化情况,记录仔鼠完成各项生理发育指标的达标日龄,采用平面翻正反射、负性趋地反射、悬吊实验、悬崖回避反射及感官反射5项指标检测仔鼠神经发育;检测母鼠血清叶酸、血浆同型半胱氨酸(homocysteine,Hcy)、SAM与S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)含量;检测仔鼠血清及脑组织叶酸,脑组织Hcy、SAM、SAH水平及DNA甲基转移酶(DNA methyltransferases,DNMTs)活性及总DNA甲基化水平,并检测仔鼠脑组织CDK5、TLR4启动子甲基化与表达水平。结果孕期叶酸干预对雌性子代幼年大鼠的早期体重具有一定的影响(P0.05),对子代幼年大鼠的早期生理发育各项指标差异均无统计学意义(P0.05);孕期叶酸缺乏可使子代幼年大鼠平面翻正反射、负性趋地反射、悬吊实验、悬崖回避反射及感官反射等神经反射行为出现延迟,叶酸补充可改善其表现。孕期叶酸补充可提高子代新生鼠血清与脑组织中的叶酸含量,降低其脑组织中Hcy,SAH含量,升高SAM/SAH比值;孕期叶酸缺乏则降低仔鼠脑组织叶酸含量、提高了仔鼠脑组织中Hcy、SAH含量,降低了SAM/SAH比值(P0.05)。孕期叶酸补充可提高仔鼠脑组织总DNMTs活性及其m RNA与蛋白表达,提高DNA总甲基化水平;孕期叶酸缺乏则降低总DNMTs活性及其表达,降低DNA总甲基化水平(P0.05)。孕期叶酸补充可以提高CDK5、TLR4基因启动子区甲基化水平,从而下调其表达;孕期叶酸缺乏则降低了CDK5、TLR4基因启动子区甲基化水平,促进其表达(P0.05)。结论本研究结果显示孕期叶酸补充可改善子代幼年大鼠神经行为学发育。机制研究提示孕期叶酸补充能够提高子代大鼠血清及脑组织中叶酸、SAM水平,降低Hcy、SAH水平,提高子代大鼠脑组织甲基化潜能及DNMTs活性和总甲基化水平。孕期叶酸补充能够上调子代脑组织CDK5、TLR4基因启动子区甲基化水平,使其发生静默,降低其表达水平。孕期叶酸补充调节CDK5、TLR4基因甲基化改善子代幼年大鼠神经行为发育。
[Abstract]:Objective the relationship between the maternal nutrition status and the offspring development during pregnancy, folic acid as a single carbon carrier can participate in various methylation reactions in the body, and DNA methylation plays an important role in the development of the neural system of the offspring, but whether the intervention of folic acid during pregnancy through DNA methylation through the modulation of modulated nerve development and its effect Specific gene loci are not yet clear. This study is intended to explore whether the level of folic acid nutrition during pregnancy can improve the neurodevelopment of young rats by modulating cyclin dependent kinase 5 (cyclin-dependent kinase 5, CDK5) and Toll like receptor 4 (Toll-like receptors 4, TLR4) during pregnancy. Methods 40 SPF grade female Sprague-Dawley (SD) rats were randomly divided into 4 groups: (1) folate deficiency group (folate-deficient diet group, DD) was fed with folic acid deficiency diet; (2) the normal folic acid group (folate-normal diet group, ND) was fed with Ye Suanzheng constant feed; (3) short term folic acid supplementation group (3) D-S) supplemented with folic acid for 10 days during the mating period and the rest of the time with folic acid normal feed; (4) the whole course of folic acid supplementation after pregnancy (folate-added diet long-period group, FD-L) was fed with folic acid supplementation. Pregnant rats were given birth as a newborn rat first days after birth (Postnatal day 1, PD1), in PD1-PD14, daily prison. The body weight changes of the offspring were measured, and the average age of the offspring was recorded, and 5 indexes were detected by plane reversal reflex, negative chemotactic reflex, suspension test, cliff avoidance reflex and sensory reflex, and serum folic acid, plasma homocysteine (homocysteine, Hcy), SAM and S- adenosine were detected. S-adenosylhomocysteine (SAH) content; detection of serum and brain tissue folic acid, Hcy, SAM, SAH level and DNA methyltransferase (DNA methyltransferases, DNMTs) activity and total DNA methylation level of the brain tissue, and detect the CDK5 of the brain tissue of the offspring and the level of methylation and expression of the TLR4 promoter. Results during pregnancy, folic acid intervened in the female. The early body weight of the young rats had a certain influence (P0.05), and there was no significant difference in the early physiological development indexes of the young adult rats (P0.05). Folic acid supplementation could improve its performance. Folic acid supplementation during pregnancy could increase the content of folic acid in serum and brain tissue of newborn rats, decrease the content of Hcy and SAH in brain tissue, increase the ratio of SAM/SAH, and decrease the content of folic acid in the brain tissue of the offspring, increase the content of Hcy and SAH in the brain tissue of the offspring, and reduce the ratio of SAM/SAH (P0.05). During pregnancy, folic acid supplementation could improve the total DNMTs activity of the brain tissue and the expression of M RNA and protein, increase the total methylation level of DNA, decrease the total DNMTs activity and its expression and decrease the total DNA methylation level (P0.05) during pregnancy, and the folic acid supplementation during pregnancy can improve the methylation level of CDK5, TLR4 gene promoter region and down regulation of the expression of the folic acid supplementation during pregnancy. Folic acid deficiency during pregnancy reduces the level of methylation in the promoter region of CDK5 and TLR4 genes and promotes its expression (P0.05). Conclusion the results of this study suggest that folic acid supplementation during pregnancy can improve the neurobehavioral development of juvenile rats. The mechanism study suggests that folic acid supplementation during pregnancy can improve the level of folic acid, SAM, and decrease Hcy, S in the serum and brain tissue of the offspring of the offspring. AH level, improve the methylation potential and DNMTs activity and total methylation level of the rat brain tissue. During pregnancy, folic acid supplementation can improve the level of methylation of CDK5, TLR4 gene promoter region, make it silent and reduce its expression level. During pregnancy, folic acid supplementation is supplemented by CDK5, TLR4 gene methylation improves the neurologic of young offspring of offspring. For development.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R715.3

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