本文關(guān)鍵詞：氧化應(yīng)激和DNA甲基化在同型半胱氨酸誘導(dǎo)神經(jīng)管畸形中的作用機(jī)制探討　出處：《北京協(xié)和醫(yī)學(xué)院》2010年博士論文　論文類型：學(xué)位論文

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【摘要】：背景：同型半胱氨酸(homocysteine, HCY),是一種含硫氨基酸,是一碳代謝的中間產(chǎn)物。神經(jīng)管畸形(Neural Tube Defects NTDs)是指胚胎發(fā)育早期,由于遺傳因素和環(huán)境因素的影響,致神經(jīng)管的發(fā)生和分化紊亂而出現(xiàn)的人類出生缺陷中最常見(jiàn)和最嚴(yán)重的一組畸形。近年來(lái)的研究表明：體內(nèi)HCY升高與NTDs關(guān)系密切,但HCY致NTDs的分子機(jī)制尚不完全清楚。 目的：研究HCY導(dǎo)致NTDs的新的致病機(jī)制,并針對(duì)致病機(jī)制提出保護(hù)措施,為神經(jīng)管畸形的治療和預(yù)防提供基礎(chǔ)。 方法：以雞胚為動(dòng)物模型,研究HCY對(duì)雞胚神經(jīng)管發(fā)育的影響及可能的分子機(jī)制。通過(guò)石蠟切片和H-E染色分析HCY對(duì)雞胚神經(jīng)管的影響；檢測(cè)HCY對(duì)活性氧(ROS)、丙二醛(MDA)、總谷胱甘肽(GSSG)、超氧化物岐化酶(SOD)、谷胱甘肽過(guò)氧化物酶(GPX)等氧化應(yīng)激相關(guān)指標(biāo)的影響；高效液相色譜(HPLC)、全胚免疫熒光、、western blot檢測(cè)HCY對(duì)DNA甲基轉(zhuǎn)移能力的影響；Taqman real timePCR檢測(cè)HCY處理后miR-124的變化；免疫組化分析SCP1.、Pax6/和Tuj1蛋白的表達(dá)；通過(guò)雞胚卵內(nèi)電轉(zhuǎn)對(duì)miR-124進(jìn)行敲低和過(guò)表達(dá),分析其對(duì)神經(jīng)管發(fā)育的影響。 結(jié)果：(1) HCY能顯著降低雞胚的生存能力,影響胚胎外血管發(fā)育,導(dǎo)致脊柱裂畸形,與抗氧化劑N-乙酰半胱氨酸(NAC)或膽堿(CHO)聯(lián)合處理能部分恢復(fù)以上表型,表明HCY導(dǎo)致的脊柱裂與氧化應(yīng)激和一碳代謝異常有關(guān)。 (2) HCY導(dǎo)致ROS明顯增加,MDA含量明顯增加,Mn-SOD活性和GPX活性明顯下降,與NAC聯(lián)合處理能部分恢復(fù)以上改變。 (3) HCY導(dǎo)致基因組DNA整體低甲基化,S-腺苷甲硫氨酸(SAM)含量下降,S-腺苷同型半胱氨酸(SAH)含量上升,SAM/SAH下降,DNMT1和DNMT3a蛋白表達(dá)下降,CHO與HCY聯(lián)合處理能部分恢復(fù)以上改變。 (4) NAC與HCY聯(lián)合處理能部分恢復(fù)HCY導(dǎo)致的基因組整體低甲基化和DNMT1及DNMT3a蛋白表達(dá)的下降,SAM、SAH及SAM/SAH則沒(méi)有明顯差別。 (5) HCY導(dǎo)致miR-124表達(dá)明顯下調(diào),與去甲基化藥物5-氮雜脫氧胞苷(5-AZA)聯(lián)合處理能部分恢復(fù)表達(dá),表明miR-124的調(diào)控與啟動(dòng)子區(qū)超甲基化有關(guān)。免疫組化顯示miR-124調(diào)控的靶基因SCP1表達(dá)上調(diào),神經(jīng)前體細(xì)胞marker Pax6表達(dá)上調(diào),神經(jīng)元marker Tuj 1表達(dá)下調(diào)。 (6)對(duì)miR-124進(jìn)行敲低后雞胚出現(xiàn)脊柱裂,SCP1和Pax6表達(dá)上調(diào),Tuj1表達(dá)下調(diào),表明miR-124在神經(jīng)管正常發(fā)育中起著重要作用。 結(jié)論：(1)本研究首次證實(shí)HCY可通過(guò)氧化應(yīng)激影響DNA甲基轉(zhuǎn)移能力,并證實(shí)NAC和CHO對(duì)HCY導(dǎo)致的NTDs具有保護(hù)作用。 (2)HCY通過(guò)氧化應(yīng)激和干擾甲基代謝影響DNA甲基轉(zhuǎn)移能力,導(dǎo)致基因組DNA整體低甲基化,并伴隨局部區(qū)域的超甲基化。這種表觀遺傳修飾可能是HCY導(dǎo)致的miR-124表達(dá)下調(diào)的原因。 (3)miR-124下調(diào)會(huì)導(dǎo)致其靶基因SCP1表達(dá)上調(diào),神經(jīng)前體細(xì)胞增多,導(dǎo)致神經(jīng)上皮增厚,可能是神經(jīng)管無(wú)法正常閉合的原因。HCY可能通過(guò)表觀遺傳修飾下調(diào)miR-124來(lái)導(dǎo)致神經(jīng)管畸形。
[Abstract]:Background: homocysteine (homocysteine, HCY), is a sulfur-containing amino acid, the intermediate product of one carbon metabolism. Neural tube defects (Neural Tube Defects NTDs) refers to the early embryo development, due to the influence of genetic and environmental factors, caused by the occurrence and differentiation of neural tube disorders and the emergence of human birth defects the most common and most serious group of malformations. Recent studies have shown that the relationship between the body increased HCY and NTDs close, but the molecular mechanism of NTDs induced by HCY is not fully clear.
Objective: To study the new pathogenesis of NTDs caused by HCY, and to provide protective measures for the pathogenesis, and provide the basis for the treatment and prevention of neural tube malformation.
Methods: the animal model for the study of embryo, HCY effects on chick neural tube development and its possible molecular mechanism. To analyze the effect of HCY on chick neural tube by paraffin sectioning and H-E staining; detection of HCY on reactive oxygen species (ROS), malondialdehyde (MDA), total glutathione (GSSG), superoxide dismutase (SOD), glutathione peroxidase (GPX) effects of oxidative stress indicators; high performance liquid chromatography (HPLC), whole mount immunofluorescence, Western blot detection of HCY effect on metastasis of DNA methyl miR-124 Taqman real timePCR; change detection after HCY treatment; immunohistochemical analysis of SCP1., the expression of Pax6/ and Tuj1 the chicken egg protein; by electroporation of miR-124 knockdown and overexpression, analyzes its influence on the development of the neural tube.
Results: (1) HCY can significantly reduce the embryo viability, affect embryonic vascular development, cause spina bifida malformation, and antioxidants of N- acetylcysteine (NAC) and choline (CHO) combined treatment can partially restore the above phenotype, suggesting that HCY causes spina bifida associated with metabolic abnormalities in oxidative stress and a carbon.
(2) HCY resulted in a significant increase in ROS, a significant increase in the content of MDA, and a significant decrease in the activity of Mn-SOD and the activity of GPX. The combined treatment with NAC could partly restore the above changes.
(3) HCY resulted in the overall hypomethylation of genomic DNA, the decrease of S- adenosylmethionine (SAM), the increase of S- adenosine homocysteine (SAH), the decrease of SAM/SAH, the decrease of DNMT1 and DNMT3a protein expression, and the combination of CHO and HCY could partially restore the above changes.
(4) combined treatment of NAC and HCY partially restored the overall hypomethylation of HCY and the decrease of DNMT1 and DNMT3a protein expression. SAM, SAH and SAM/SAH showed no significant difference.
(5) HCY induced down-regulation of miR-124 expression, and demethylation agent 5- aza deoxycytidine (5-AZA) combined treatment can partially restore the expression, regulation and show that the miR-124 promoter hypermethylation. Immunohistochemistry showed that the expression of SCP1 target genes regulated by miR-124, the neuronal precursor cells marker Pax6 expression upregulation of neuronal marker downregulation of Tuj 1 expression.
(6) after the knockdown of miR-124, the chick embryo appeared spina bifida, the expression of SCP1 and Pax6 was up-regulated, and the expression of Tuj1 was down regulated, indicating that miR-124 plays an important role in the normal development of neural tube.
Conclusions: (1) this study is the first to confirm that HCY can affect DNA methyltransferability through oxidative stress, and that NAC and CHO have protective effects on NTDs induced by HCY.
(2) HCY affects DNA methyltransferability through oxidative stress and interference with methyl metabolism, resulting in the overall hypomethylation of genomic DNA and the hypermethylation of the local region. This epigenetic modification may be the reason for the downregulation of miR-124 expression induced by HCY.
(3) down regulation of miR-124 will result in the up regulation of SCP1 expression of target genes, the increase of neural precursor cells, resulting in thickening of neural epithelium, which may be the reason why neural tube can not normally shut down..HCY may cause miR-124 through down regulation of epigenetic modification.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王錦;硒和維生素E緩解肉雞氧化應(yīng)激的研究[D];西北農(nóng)林科技大學(xué);2012年

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本文編號(hào)：1386499