發(fā)布時間：2018-06-16 08:23

  本文選題：海馬 + 甜菜堿�。� 參考：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：海馬是腦內(nèi)重要的組成成分,與記憶、應(yīng)激、能量代謝等有著密切的關(guān)系。海馬可塑性強且對發(fā)育早期的環(huán)境敏感。充足的甲基供體供應(yīng)對腦的發(fā)育很重要。眾所周知,甜菜堿因為含有三個活性甲基是高效的甲基供體。在大鼠模型上,有研究表明妊娠期補充甲基供體(膽堿)能夠促進子代海馬細胞的增殖,抑制海馬細胞的凋亡,但具體機制尚不清楚。本文的目的是探究母體補充甲基供體甜菜堿對子代海馬細胞的影響,以及產(chǎn)生影響的具體機制。本文首先以新生仔豬為研究對象揭示妊娠母豬日糧添加甜菜堿對雄性新生仔豬海馬內(nèi)甜菜堿/蛋氨酸代謝以及DNA甲基轉(zhuǎn)移酶表達的影響,探究甲基供體甜菜堿影響海馬細胞增殖的機制;其次我們研究了這種影響能否延續(xù)到仔豬斷奶時期;此外,我們用HT-22海馬細胞進一步研究了細胞內(nèi)的分子機制。1甜菜堿通過影響一碳代謝和DNA甲基化調(diào)節(jié)新生仔豬海馬細胞的增殖選擇12頭二元雜交母豬(長白×約克夏),進行人工授精(混合好的杜洛克公豬的精子),受精后的母豬被隨機平均分成對照組和試驗組兩組。二元雜交母豬配種前一個月飼喂標(biāo)準(zhǔn)日糧,配種后對照組飼喂標(biāo)準(zhǔn)日糧,試驗組飼喂添加甜菜堿(3 g/kg)的日糧,一天飼喂3次,并自由飲水,直至分娩結(jié)束。等到母豬分娩后,我們選擇平均體重的雄性仔豬(每組六頭)采集血液和海馬樣品。將海馬樣品裝入標(biāo)記好的2 mL的EP管內(nèi),然后迅速投入液氮中,之后轉(zhuǎn)移至-75℃保存。血液完全凝固后,離心取血清,于-20℃保存。實驗發(fā)現(xiàn),妊娠母豬飼喂甜菜堿沒有改變新生仔豬體重、海馬重、海馬重/體重,但是增加了海馬DNA含量以及增殖相關(guān)基因mRNA的表達。飼喂甜菜堿組顯著增加新生仔豬血液內(nèi)蛋氨酸的含量(P0.05)并顯著上調(diào)新生仔豬海馬內(nèi)BHMT、GNMT和DNMT1的mRNA和蛋白的表達。在甜菜堿組新生仔豬海馬內(nèi)胰島素樣生長因子2(IGF2)和它的受體IGF1R和IGF2R的表達都顯著增加(P0.05),同時伴隨著下游信號通路ERK1/2的激活(P0.01),此外IGF2的差異甲基化區(qū)域DMR1和DMR2高甲基化(P0.05)。以上結(jié)果說明:妊娠母豬日糧添加甜菜堿可以促進新生仔豬海馬內(nèi)甜菜堿/蛋氨酸代謝以及DNA甲基轉(zhuǎn)移酶的表達,進而引起IGF2的DMR高甲基化并伴隨著IGF2和細胞增殖/抗凋亡的基因高表達。2甜菜堿通過影響一碳代謝和DNA甲基化調(diào)節(jié)斷奶仔豬海馬細胞的增殖母豬分娩后對照組繼續(xù)飼喂標(biāo)準(zhǔn)日糧,試驗組繼續(xù)飼喂添加甜菜堿(3 g/kg)的日糧,每天飼喂兩次,時間分別為10:00和16:00,每次飼喂的量相等,自由飲水。仔豬在28日齡斷奶采樣。我們選擇平均體重的雄性仔豬(每組六頭)采集血液和海馬樣品。將海馬樣品裝入標(biāo)記好的2 mL的EP管內(nèi),然后迅速投入液氮中,之后轉(zhuǎn)移至-75℃保存。血液完全凝固后,離心取血清,于-20℃保存。實驗發(fā)現(xiàn):妊娠母豬飼喂甜菜堿顯著增加了斷奶仔豬血液內(nèi)甜菜堿的含量(P0.05),并下調(diào)斷奶仔豬海馬內(nèi)BHMT的蛋白(P0.05)表達。但是,妊娠母豬飼喂甜菜堿沒有改變斷奶仔豬體重、海馬重、海馬重/體重和日增重,也沒有改變仔豬海馬的DNA含量以及增殖相關(guān)基因的mRNA表達以及基因組DNA甲基化水平。以上結(jié)果說明,妊娠母豬日糧添加甜菜堿對新生仔豬海馬細胞增殖的影響沒有延續(xù)到斷奶仔豬。3甲基供體影響海馬細胞增殖的機制高低劑量甜菜堿對海馬細胞BHMT蛋白表達的影響甜菜堿對新生仔豬和斷奶仔豬的影響不同。我們推測這種差異可能是由于兩個時期內(nèi)血液中甜菜堿的含量不同引起代謝過程中關(guān)鍵酶BHMT的表達不同造成的。進而我們設(shè)計了一個小實驗來研究高低劑量甜菜堿對海馬細胞BHMT蛋白表達的影響。我們將HT-22小鼠海馬細胞培養(yǎng)在DMEM + 10%FBS的培養(yǎng)基中。試驗共分3組:(1)空白組(Con);(2)低劑量甜菜堿組(1μM甜菜堿)(LB);(3)高劑量甜菜堿組(1mM甜菜堿)(HB);在細胞生長到70%時更換培養(yǎng)液,按照分組分別加入不同的培養(yǎng)基培養(yǎng)24 h;之后棄掉培養(yǎng)基收細胞。實驗發(fā)現(xiàn):高低劑量甜菜堿對細胞活力以及BHMT的蛋白表達均沒有影響。暗示著造成新生仔豬和斷奶仔豬結(jié)果不同的原因可能是甜菜堿沒有直接發(fā)揮作用,而是通過影響葉酸的代謝途徑發(fā)揮作用。葉酸影響海馬細胞增殖的機制采用小鼠海馬神經(jīng)細胞系(HT-22)進行培養(yǎng),試驗共分2組:(1)對照組(Con)(2)葉酸缺乏組(FD);在細胞生長到70%時更換培養(yǎng)液,按照分組分別加入不同的培養(yǎng)基培養(yǎng)24 h;之后吸掉培養(yǎng)基,收細胞。實驗發(fā)現(xiàn):培養(yǎng)基中葉酸缺乏可以顯著降低細胞活力(P0.05),降低海馬細胞DNA含量和增殖相關(guān)基因的表達以及培養(yǎng)基中IGF1的含量。卻顯著上調(diào)細胞內(nèi)BHMT、MAT2b和DNMT3a的蛋白表達。說明葉酸對海馬細胞的增殖很重要,不僅可以影響蛋氨酸代謝通路,也可以影響增殖相關(guān)因子IGF1。綜上所述:妊娠母豬日糧添加甜菜堿可以促進新生仔豬海馬內(nèi)甜菜堿/蛋氨酸代謝以及DNA甲基轉(zhuǎn)移酶的表達,進而引起IGF2的DMR高甲基化并伴隨著IGF2和細胞增殖/抗凋亡的基因高表達。但是這種影響并不能持續(xù)到斷奶時期。我們探究其中可能的機制,推測這種現(xiàn)象是由于血液內(nèi)甜菜堿含量高低不同造成的。為此,我們做了一個小實驗,結(jié)果顯示培養(yǎng)基中高低劑量甜菜堿對海馬細胞的BHMT表達沒有影響。結(jié)果暗示,造成新生仔豬和斷奶仔豬結(jié)果不同的原因可能是甜菜堿沒有直接發(fā)揮作用,而是通過影響葉酸的代謝途徑發(fā)揮作用。我們用缺乏葉酸的培養(yǎng)基處理海馬細胞,發(fā)現(xiàn)葉酸缺乏不僅可以影響蛋氨酸代謝通路,也可以影響增殖相關(guān)因子IGF1的表達。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S828

【參考文獻】

相關(guān)期刊論文 前1條

1 Anjana Munshi;Shanti Duvvuri;;基因組印記研究進展(英文)[J];遺傳學(xué)報;2007年02期

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本文編號：2026034