氧化应激和DNA甲基化在同型半胱氨酸诱导神经管畸形中的作用机制探讨

发布时间：2018-01-06 05:28

本文关键词：氧化应激和DNA甲基化在同型半胱氨酸诱导神经管畸形中的作用机制探讨　出处：《北京协和医学院》2010年博士论文　论文类型：学位论文

【摘要】：背景：同型半胱氨酸(homocysteine, HCY),是一种含硫氨基酸,是一碳代谢的中间产物。神经管畸形(Neural Tube Defects NTDs)是指胚胎发育早期,由于遗传因素和环境因素的影响,致神经管的发生和分化紊乱而出现的人类出生缺陷中最常见和最严重的一组畸形。近年来的研究表明：体内HCY升高与NTDs关系密切,但HCY致NTDs的分子机制尚不完全清楚。目的：研究HCY导致NTDs的新的致病机制,并针对致病机制提出保护措施,为神经管畸形的治疗和预防提供基础。方法：以鸡胚为动物模型,研究HCY对鸡胚神经管发育的影响及可能的分子机制。通过石蜡切片和H-E染色分析HCY对鸡胚神经管的影响；检测HCY对活性氧(ROS)、丙二醛(MDA)、总谷胱甘肽(GSSG)、超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GPX)等氧化应激相关指标的影响；高效液相色谱(HPLC)、全胚免疫荧光、、western blot检测HCY对DNA甲基转移能力的影响；Taqman real timePCR检测HCY处理后miR-124的变化；免疫组化分析SCP1.、Pax6/和Tuj1蛋白的表达；通过鸡胚卵内电转对miR-124进行敲低和过表达,分析其对神经管发育的影响。结果：(1) HCY能显著降低鸡胚的生存能力,影响胚胎外血管发育,导致脊柱裂畸形,与抗氧化剂N-乙酰半胱氨酸(NAC)或胆碱(CHO)联合处理能部分恢复以上表型,表明HCY导致的脊柱裂与氧化应激和一碳代谢异常有关。 (2) HCY导致ROS明显增加,MDA含量明显增加,Mn-SOD活性和GPX活性明显下降,与NAC联合处理能部分恢复以上改变。 (3) HCY导致基因组DNA整体低甲基化,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蛋白表达下降,CHO与HCY联合处理能部分恢复以上改变。 (4) NAC与HCY联合处理能部分恢复HCY导致的基因组整体低甲基化和DNMT1及DNMT3a蛋白表达的下降,SAM、SAH及SAM/SAH则没有明显差别。 (5) HCY导致miR-124表达明显下调,与去甲基化药物5-氮杂脱氧胞苷(5-AZA)联合处理能部分恢复表达,表明miR-124的调控与启动子区超甲基化有关。免疫组化显示miR-124调控的靶基因SCP1表达上调,神经前体细胞marker Pax6表达上调,神经元marker Tuj 1表达下调。 (6)对miR-124进行敲低后鸡胚出现脊柱裂,SCP1和Pax6表达上调,Tuj1表达下调,表明miR-124在神经管正常发育中起着重要作用。结论：(1)本研究首次证实HCY可通过氧化应激影响DNA甲基转移能力,并证实NAC和CHO对HCY导致的NTDs具有保护作用。 (2)HCY通过氧化应激和干扰甲基代谢影响DNA甲基转移能力,导致基因组DNA整体低甲基化,并伴随局部区域的超甲基化。这种表观遗传修饰可能是HCY导致的miR-124表达下调的原因。 (3)miR-124下调会导致其靶基因SCP1表达上调,神经前体细胞增多,导致神经上皮增厚,可能是神经管无法正常闭合的原因。HCY可能通过表观遗传修饰下调miR-124来导致神经管畸形。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

【引证文献】