神经管畸形鼠中叶酸干预与H3K27me3表达关系的研究

发布时间：2018-02-04 20:39

  本文关键词： 叶酸 神经管畸形 全反式维甲酸 H3K27me3 Adcy2　出处：《宁夏医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的以全反式维甲酸(All-trans Retinoic Acid,ATRA)诱导构建的神经管畸形鼠模型为研究对象,探索在神经管畸形(Neural Tube Defects,NTDs)的发生发展过程中H3K27me3(H3组蛋白第27位赖氨酸的三甲基化状态)和其下游叶酸代谢相关基因Adcy2的表达变化情况,了解叶酸干预在这一过程中的影响作用,探讨在神经管畸形的发生过程中叶酸干预的预防作用,为防治神经管畸形(Neural Tube Defects,NTDs)的早期发生提供可靠的理论依据。方法1.动物模型的建立:把24只健康且性成熟的雌性SD大鼠分为随机三组;1.1胎鼠大体形态学观察:于E15d时剖宫取胎,观察胎鼠的形态学特征,记录各个类型胎鼠数量,统计并分析畸形胎鼠发生率。1.2胎鼠神经管电镜下观察:剥离出各组胎鼠神经管,经过电镜包埋后置于透射电子显微镜下观察并拍照。1.3病理学观察:取各组胎鼠组织进行HE染色,并在显微镜下观察其病理学变化。2.各组胎鼠神经管中H3K27me3表达量的检测:采用免疫组织化学染色法和Western blot技术检测三组胎鼠中的H3K27me3的表达,并对其进行定性、定量分析。3.各组胎鼠神经管中基因Acdy2表达量的测定:采用QRT-PCR技术对各组胎鼠中Adcy2基因的表达量进行测定分析。结果1.动物模型制作及观察结果:1.1统计结果分析显示正常组未发现畸形胎鼠,实验组的神经管畸形发生率为84%,叶酸干预组的畸形发生率为41.1%;实验组畸形发生率和正常组比较明显升高,差异具有统计学意义(P0.01),叶酸干预组比正常组畸形率增加,但与实验组比较其畸形率显著下降,差异具有统计学意义(P0.05)。1.2各组神经管电镜下观察结果:正常组神经管组织,其细胞核完整,呈圆形或卵圆形。线粒体清晰可见;实验组神经管组织细胞核不完整,变形严重,线粒体出现皱缩,甚至溶解;叶酸干预组神经管细胞核虽然也存在变形、皱缩现象,但与实验组比较,其状况有相应的改善,细胞间隙趋于正常,少部分线粒体出现皱缩、溶解。1.3 HE染色结果:正常组胎鼠可看到其神经管发育正常,脊髓形态正常。实验组中显性脊柱裂的胎鼠,其脊髓改变复杂,脊柱背侧覆皮中断不连续,脊髓结构破坏紊乱,脊髓伴随脊膜向外膨出,多伴有炎性细胞侵袭。叶酸干预组中脊柱裂较少,无尾畸形较多,相比实验组而言叶酸干预组发育有所改善,但比正常组发育仍然较差。2.各组神经管中H3K27me3表达量的检测:免疫组化结果显示:正常组中H3K27me3的表达量低,实验组中为强阳性表达,叶酸干预组中其表达量稍低于实验组,但并没有显示出差异性,而与正常组相比较,差异较明显,具有统计学意义(P0.05)。Western blot检测结果显示:实验组中H3K27me3的表达量高于正常组和叶酸干预组,差异有统计学意义(P0.05),叶酸干预组表达量介于两组之间,差异明显,具有统计学意义(P0.05)。3.各组神经管中Adcy2基因的QRT-PCR检测结果显示:在实验组中高表达,与正常组和叶酸干预组比较,差异具有统计学意义(P0.05);在叶酸干预组中的表达量介于两组之间,差异具有统计学意义(P0.05)。结论1.叶酸干预,可以有效降低神经管畸形率的发生;2.神经管畸形胎鼠中,H3K27me3表达水平上调,叶酸代谢相关基因Adcy2的表达上调,叶酸干预可以有效降低两者的表达量。
[Abstract]:The purpose of all trans retinoic acid (All-trans Retinoic Acid, ATRA) induced by construction of neural tube defects in rat model as the research object, to explore in neural tube defects (Neural Tube, Defects, NTDs) in the process of the occurrence and development of H3K27me3 (histone H3 at lysine twenty-seventh trimethylation state) and expression of Adcy2 the downstream genes of folate metabolism, to understand the effect of folic acid in this process, to investigate the preventive effect of folic acid in the process of occurrence of malformations of the neural tube, for the prevention of neural tube defects (Neural Tube, Defects, NTDs) provide a reliable theoretical basis for the early occurrence. Methods 1. animal model: 24 healthy and mature female SD rats were randomly divided into three groups; 1.1 fetal morphological observation: in E15d cesarean section, observe the morphological characteristics of fetal rats, record the number of each type of fetal rats, and statistical analysis of abnormal The incidence of fetal rat shaped nerve.1.2 fetal rat tube under electron microscopy, stripping the nerve in all groups after embedding tube, electron microscope in transmission electron microscope observation and pathological observation:.1.3 pictures were taken and fetal tissue HE staining to observe the pathological changes and neural tube of H3K27me3 fetal rats in.2. groups to detect the expression under the microscope: the immunohistochemical staining method and Western blot expression in fetal rats in three groups in H3K27me3, and its qualitative, quantitative analysis of.3. in all groups of neural tube of Acdy2 expression: the expression of Adcy2 was determined by each gene in fetal rats with QRT-PCR technology were analyzed. Results 1. animal model and observation results: 1.1 statistical analysis showed that the normal group found no abnormalities of fetal rats in experimental group, the incidence of neural tube defects was 84%, folic acid intervention group malformation rate was 41 .1%; experimental group and deformity rate increased significantly compared to normal group, the difference was statistically significant (P0.01), increased folic acid group than the normal group but the deformity rate between experimental group and the malformation rate was significantly decreased, the difference was statistically significant (P0.05).1.2 groups of neural tube observation under electron microscope: normal group neural tube tissue the nucleus, complete, rounded or oval. Mitochondria visible; the experimental group the neural tube tissue nucleus is not complete, severe deformation, mitochondria shrinkage, even dissolution; folic acid intervention group neural tube nucleus although there is deformation, collapse, but compared with the experimental group, the situation has improved accordingly, the cell gap tends to normally, a few mitochondria were shrunken, dissolved.1.3 HE staining results: normal fetal rats can see the development of the neural tube normal, normal morphology of spinal cord. Fetal spina bifida in the experimental group, the ridge The complex changes of dorsal spinal cord, overlying skin disruption is not continuous, destroying the structure of spinal cord disorders, with spinal dural bulge outward, accompanied by inflammatory cell invasion. Folic acid in the intervention group of spina bifida is less, no tail deformity is more, compared to the experimental group for folic acid group development has improved, but still less than the normal growth of.2. the expression of H3K27me3 in neural tube detection: the results of immunohistochemistry showed that the expression of H3K27me3 in the normal group was low in the experimental group, strong positive expression, the expression of folic acid intervention group is slightly lower than the experimental group, but did not show the opposite, compared with the normal group, the difference was significant, with statistical significance (P0.05).Western blot assay showed that the expression of H3K27me3 in experimental group was higher than that of normal group and folic acid intervention group, the difference was statistically significant (P0.05), the expression amount is between folic acid intervention group between the two groups, significant difference , with statistical significance (P0.05) detection results of QRT-PCR.3. groups in neural tube of Adcy2 gene showed high expression in the experimental group compared with the normal group and folic acid intervention group, the difference was statistically significant (P0.05); in the folic acid group and the expression levels between two groups, the difference was statistically significant (P0.05 1.). Conclusion the folic acid intervention, can effectively reduce the incidence of neural tube defects; 2. of neural tube defects in fetal rats, H3K27me3 expression level increased, upregulation of folate metabolism related gene Adcy2 expression, folic acid can effectively reduce the expression of the two.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R715.3
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本文编号：1491086