甲基供体通过影响一碳代谢和DNA甲基化调节海马细胞的增殖

发布时间：2018-06-16 08:23

本文选题：海马 + 甜菜碱　；参考：《南京农业大学》2015年硕士论文

【摘要】：海马是脑内重要的组成成分,与记忆、应激、能量代谢等有着密切的关系。海马可塑性强且对发育早期的环境敏感。充足的甲基供体供应对脑的发育很重要。众所周知,甜菜碱因为含有三个活性甲基是高效的甲基供体。在大鼠模型上,有研究表明妊娠期补充甲基供体(胆碱)能够促进子代海马细胞的增殖,抑制海马细胞的凋亡,但具体机制尚不清楚。本文的目的是探究母体补充甲基供体甜菜碱对子代海马细胞的影响,以及产生影响的具体机制。本文首先以新生仔猪为研究对象揭示妊娠母猪日粮添加甜菜碱对雄性新生仔猪海马内甜菜碱/蛋氨酸代谢以及DNA甲基转移酶表达的影响,探究甲基供体甜菜碱影响海马细胞增殖的机制;其次我们研究了这种影响能否延续到仔猪断奶时期;此外,我们用HT-22海马细胞进一步研究了细胞内的分子机制。1甜菜碱通过影响一碳代谢和DNA甲基化调节新生仔猪海马细胞的增殖选择12头二元杂交母猪(长白×约克夏),进行人工授精(混合好的杜洛克公猪的精子),受精后的母猪被随机平均分成对照组和试验组两组。二元杂交母猪配种前一个月饲喂标准日粮,配种后对照组饲喂标准日粮,试验组饲喂添加甜菜碱(3 g/kg)的日粮,一天饲喂3次,并自由饮水,直至分娩结束。等到母猪分娩后,我们选择平均体重的雄性仔猪(每组六头)采集血液和海马样品。将海马样品装入标记好的2 mL的EP管内,然后迅速投入液氮中,之后转移至-75℃保存。血液完全凝固后,离心取血清,于-20℃保存。实验发现,妊娠母猪饲喂甜菜碱没有改变新生仔猪体重、海马重、海马重/体重,但是增加了海马DNA含量以及增殖相关基因mRNA的表达。饲喂甜菜碱组显著增加新生仔猪血液内蛋氨酸的含量(P0.05)并显著上调新生仔猪海马内BHMT、GNMT和DNMT1的mRNA和蛋白的表达。在甜菜碱组新生仔猪海马内胰岛素样生长因子2(IGF2)和它的受体IGF1R和IGF2R的表达都显著增加(P0.05),同时伴随着下游信号通路ERK1/2的激活(P0.01),此外IGF2的差异甲基化区域DMR1和DMR2高甲基化(P0.05)。以上结果说明:妊娠母猪日粮添加甜菜碱可以促进新生仔猪海马内甜菜碱/蛋氨酸代谢以及DNA甲基转移酶的表达,进而引起IGF2的DMR高甲基化并伴随着IGF2和细胞增殖/抗凋亡的基因高表达。2甜菜碱通过影响一碳代谢和DNA甲基化调节断奶仔猪海马细胞的增殖母猪分娩后对照组继续饲喂标准日粮,试验组继续饲喂添加甜菜碱(3 g/kg)的日粮,每天饲喂两次,时间分别为10:00和16:00,每次饲喂的量相等,自由饮水。仔猪在28日龄断奶采样。我们选择平均体重的雄性仔猪(每组六头)采集血液和海马样品。将海马样品装入标记好的2 mL的EP管内,然后迅速投入液氮中,之后转移至-75℃保存。血液完全凝固后,离心取血清,于-20℃保存。实验发现:妊娠母猪饲喂甜菜碱显著增加了断奶仔猪血液内甜菜碱的含量(P0.05),并下调断奶仔猪海马内BHMT的蛋白(P0.05)表达。但是,妊娠母猪饲喂甜菜碱没有改变断奶仔猪体重、海马重、海马重/体重和日增重,也没有改变仔猪海马的DNA含量以及增殖相关基因的mRNA表达以及基因组DNA甲基化水平。以上结果说明,妊娠母猪日粮添加甜菜碱对新生仔猪海马细胞增殖的影响没有延续到断奶仔猪。3甲基供体影响海马细胞增殖的机制高低剂量甜菜碱对海马细胞BHMT蛋白表达的影响甜菜碱对新生仔猪和断奶仔猪的影响不同。我们推测这种差异可能是由于两个时期内血液中甜菜碱的含量不同引起代谢过程中关键酶BHMT的表达不同造成的。进而我们设计了一个小实验来研究高低剂量甜菜碱对海马细胞BHMT蛋白表达的影响。我们将HT-22小鼠海马细胞培养在DMEM + 10%FBS的培养基中。试验共分3组:(1)空白组(Con);(2)低剂量甜菜碱组(1μM甜菜碱)(LB);(3)高剂量甜菜碱组(1mM甜菜碱)(HB);在细胞生长到70%时更换培养液,按照分组分别加入不同的培养基培养24 h;之后弃掉培养基收细胞。实验发现:高低剂量甜菜碱对细胞活力以及BHMT的蛋白表达均没有影响。暗示着造成新生仔猪和断奶仔猪结果不同的原因可能是甜菜碱没有直接发挥作用,而是通过影响叶酸的代谢途径发挥作用。叶酸影响海马细胞增殖的机制采用小鼠海马神经细胞系(HT-22)进行培养,试验共分2组:(1)对照组(Con)(2)叶酸缺乏组(FD);在细胞生长到70%时更换培养液,按照分组分别加入不同的培养基培养24 h;之后吸掉培养基,收细胞。实验发现:培养基中叶酸缺乏可以显著降低细胞活力(P0.05),降低海马细胞DNA含量和增殖相关基因的表达以及培养基中IGF1的含量。却显著上调细胞内BHMT、MAT2b和DNMT3a的蛋白表达。说明叶酸对海马细胞的增殖很重要,不仅可以影响蛋氨酸代谢通路,也可以影响增殖相关因子IGF1。综上所述:妊娠母猪日粮添加甜菜碱可以促进新生仔猪海马内甜菜碱/蛋氨酸代谢以及DNA甲基转移酶的表达,进而引起IGF2的DMR高甲基化并伴随着IGF2和细胞增殖/抗凋亡的基因高表达。但是这种影响并不能持续到断奶时期。我们探究其中可能的机制,推测这种现象是由于血液内甜菜碱含量高低不同造成的。为此,我们做了一个小实验,结果显示培养基中高低剂量甜菜碱对海马细胞的BHMT表达没有影响。结果暗示,造成新生仔猪和断奶仔猪结果不同的原因可能是甜菜碱没有直接发挥作用,而是通过影响叶酸的代谢途径发挥作用。我们用缺乏叶酸的培养基处理海马细胞,发现叶酸缺乏不仅可以影响蛋氨酸代谢通路,也可以影响增殖相关因子IGF1的表达。
[Abstract]:The hippocampus is an important component of the brain, which is closely related to memory, stress, and energy metabolism. The hippocampus is highly plastic and sensitive to the early development environment. Sufficient methyl donor supply is important for the development of the brain. As we all know, betaine is a highly effective methyl donor because it contains three active methyl groups. The study shows that supplemental methyl donor (choline) during pregnancy can promote the proliferation of hippocampal cells and inhibit the apoptosis of hippocampal cells, but the specific mechanism is not clear. The purpose of this article is to explore the effect of maternal supplementation of methyl donor betaine on the hippocampal cells and the specific mechanism of producing shadow. First of all, the newborn piglets were studied. Objective to reveal the effect of betaine on the metabolism of betaine / methionine and DNA methyltransferase in the hippocampus of newborn piglets, and explore the mechanism of methyl donor betaine on the proliferation of hippocampal cells. Secondly, we studied the effect of this effect on the weaning period of piglets. In addition, we use HT-22 hippocampus fine. The molecular mechanism of intracellular molecular mechanism.1 betaine was further studied through the effects of one carbon metabolism and DNA methylation on the regulation of the proliferation of newborn piglets' hippocampal cells. 12 two yuan hybrids (Changbai x Yorkshire) were selected for artificial insemination (the sperm of the mixed Duroc boar). The sows were randomly divided into the control group and the experiment after the spermatogenesis. Group two. Two yuan hybrid sows were fed a standard diet one month before breeding, and the control group was fed with standard diet. The experimental group was fed with betaine (3 g/kg) diet, feeding 3 times a day and drinking water freely until the end of childbirth. After the sow delivery, we chose the normal weight male piglets (six groups of each group) to collect blood and hippocampus. Samples were loaded into the labeled 2 mL EP tube, then quickly put into liquid nitrogen and then transferred to -75 C. After the blood completely solidified, serum was centrifuged and stored at -20 C. It was found that gestation sow feeding betaine did not change the weight of newborn piglets, hippocampus weight, hippocampus weight / weight, but increased the content of DNA in the hippocampus. And the expression of the proliferation related gene mRNA. Feeding betaine significantly increased the content of methionine in the blood of newborn piglets (P0.05) and increased the expression of mRNA and protein of BHMT, GNMT and DNMT1 in the hippocampus of newborn piglets. The expression of insulin like growth factor 2 (IGF2) and its receptor IGF1R and IGF2R in the hippocampus of the newborn piglets of the betaine group A significant increase (P0.05), accompanied by the activation of the downstream signal pathway ERK1/2 (P0.01), and the difference of IGF2 methylation region DMR1 and DMR2 hypermethylation (P0.05). The above results indicate that the addition of betaine in the pregnant sow diet can promote the metabolism of betaine / methionine and the expression of DNA methyltransferase in the hippocampus of newborn piglets, and thus cause IG. The DMR hypermethylation of F2 and the high expression of IGF2 and cell proliferation / anti apoptotic gene.2 betaine continued to feed the standard diet of the control group after the effect of one carbon metabolism and DNA methylation regulating the proliferating sow of the weanling pig's hippocampal cells. The test group continued feeding the diet supplemented with betaine (3 g/kg), feeding two times a day. At 10:00 and 16:00, the amount of feeding was equal, free drinking water. The piglets were weaned at 28 days of age. We selected the average weight male piglets (six heads of each group) to collect blood and hippocampus samples. The hippocampus samples were loaded into the labeled 2 mL EP tube and then quickly put into the liquid nitrogen and then transferred to -75 C. after the blood was completely solidified, the blood was completely solidified. The experiment found that the feeding of betaine in pregnant sows increased the content of betaine in the blood of weaned piglets (P0.05) and decreased the expression of BHMT protein (P0.05) in weanling piglets, but it did not change the weight of the weaned piglets, the weight of the hippocampus, the weight of the hippocampus, the weight of the hippocampus, and the daily weight gain, nor did the pregnant sows fed with betaine. The DNA content, the mRNA expression of the proliferation related genes and the level of genomic DNA methylation were changed. The results showed that the effects of dietary betaine on the proliferation of hippocampal cells in newborn piglets did not continue to the mechanism of.3 methyl donor of weanling piglets. The effect of betaine on the expression of BHMT protein in horse cells is different from the effects of betaine on newborn piglets and weanling piglets. We speculate that this difference may be caused by the difference in the expression of the key enzyme BHMT in the metabolic process caused by different content of betaine in the two periods. The effect of alkali on the expression of BHMT protein in hippocampal cells. We cultured HT-22 mice hippocampal cells in the medium of DMEM + 10%FBS. The test was divided into 3 groups: (1) blank group (Con); (2) low dose betaine group (1 M betaine) (LB); (3) high dose of betaine group (HB); when cells grew to 70%, the culture solution was replaced, respectively added in groups, respectively added It was found that high and low doses of betaine had no effect on cell viability and BHMT protein expression, suggesting that the result of the difference in the results of newborn piglets and weaned piglets may be that betaine did not direct volatiles, but by affecting the metabolic pathways of folic acid. The mechanism of the effect of folic acid on the proliferation of hippocampal cells was cultured in the hippocampal cell line (HT-22) in mice. The experiment was divided into 2 groups: (1) the control group (Con) (2) folate deficiency group (FD); the culture medium was replaced at 70% when the cells grew to 70%, and the culture medium was added to 24 h according to the group, then the medium was sucked out and the cell was collected. Experimental discovery was found. The folate deficiency in the medium can significantly reduce the cell activity (P0.05), reduce the DNA content of the hippocampal cells and the expression of the proliferation related genes and the content of IGF1 in the medium. However, the protein expression of BHMT, MAT2b and DNMT3a in the cells is up to be raised. It is indicated that folic acid is very important for the proliferation of hippocampus cells and not only affects the methionine metabolic pathway, but also the expression of the protein expression in the cell. IGF1. can also affect the proliferation related factor: Betaine can promote the metabolism of betaine / methionine and the expression of DNA methyltransferase in the hippocampus of newborn piglets, and then cause the DMR hypermethylation of IGF2 and the high expression of IGF2 and cell proliferation / anti apoptosis genes. During the weaning period, we explored the possible mechanism, presumably due to the different levels of betaine in the blood. To this end, we made a small experiment that showed that high and low doses of betaine did not affect the expression of BHMT in the hippocampus. Results suggested that the newborn piglets and weanling piglets were formed. The reason may be that betaine does not play a direct role, but by affecting the metabolic pathway of folic acid. We use the culture medium without folic acid to treat hippocampal cells. It is found that folate deficiency can not only affect methionine metabolic pathway, but also affect the expression of proliferation phase factor IGF1.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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