本文關(guān)鍵詞： 葉酸 神經(jīng)管畸形 全反式維甲酸 H3K27me3 Adcy2　出處：《寧夏醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的以全反式維甲酸(All-trans Retinoic Acid,ATRA)誘導(dǎo)構(gòu)建的神經(jīng)管畸形鼠模型為研究對象,探索在神經(jīng)管畸形(Neural Tube Defects,NTDs)的發(fā)生發(fā)展過程中H3K27me3(H3組蛋白第27位賴氨酸的三甲基化狀態(tài))和其下游葉酸代謝相關(guān)基因Adcy2的表達變化情況,了解葉酸干預(yù)在這一過程中的影響作用,探討在神經(jīng)管畸形的發(fā)生過程中葉酸干預(yù)的預(yù)防作用,為防治神經(jīng)管畸形(Neural Tube Defects,NTDs)的早期發(fā)生提供可靠的理論依據(jù)。方法1.動物模型的建立:把24只健康且性成熟的雌性SD大鼠分為隨機三組;1.1胎鼠大體形態(tài)學(xué)觀察:于E15d時剖宮取胎,觀察胎鼠的形態(tài)學(xué)特征,記錄各個類型胎鼠數(shù)量,統(tǒng)計并分析畸形胎鼠發(fā)生率。1.2胎鼠神經(jīng)管電鏡下觀察:剝離出各組胎鼠神經(jīng)管,經(jīng)過電鏡包埋后置于透射電子顯微鏡下觀察并拍照。1.3病理學(xué)觀察:取各組胎鼠組織進行HE染色,并在顯微鏡下觀察其病理學(xué)變化。2.各組胎鼠神經(jīng)管中H3K27me3表達量的檢測:采用免疫組織化學(xué)染色法和Western blot技術(shù)檢測三組胎鼠中的H3K27me3的表達,并對其進行定性、定量分析。3.各組胎鼠神經(jīng)管中基因Acdy2表達量的測定:采用QRT-PCR技術(shù)對各組胎鼠中Adcy2基因的表達量進行測定分析。結(jié)果1.動物模型制作及觀察結(jié)果:1.1統(tǒng)計結(jié)果分析顯示正常組未發(fā)現(xiàn)畸形胎鼠,實驗組的神經(jīng)管畸形發(fā)生率為84%,葉酸干預(yù)組的畸形發(fā)生率為41.1%;實驗組畸形發(fā)生率和正常組比較明顯升高,差異具有統(tǒng)計學(xué)意義(P0.01),葉酸干預(yù)組比正常組畸形率增加,但與實驗組比較其畸形率顯著下降,差異具有統(tǒng)計學(xué)意義(P0.05)。1.2各組神經(jīng)管電鏡下觀察結(jié)果:正常組神經(jīng)管組織,其細胞核完整,呈圓形或卵圓形。線粒體清晰可見;實驗組神經(jīng)管組織細胞核不完整,變形嚴(yán)重,線粒體出現(xiàn)皺縮,甚至溶解;葉酸干預(yù)組神經(jīng)管細胞核雖然也存在變形、皺縮現(xiàn)象,但與實驗組比較,其狀況有相應(yīng)的改善,細胞間隙趨于正常,少部分線粒體出現(xiàn)皺縮、溶解。1.3 HE染色結(jié)果:正常組胎鼠可看到其神經(jīng)管發(fā)育正常,脊髓形態(tài)正常。實驗組中顯性脊柱裂的胎鼠,其脊髓改變復(fù)雜,脊柱背側(cè)覆皮中斷不連續(xù),脊髓結(jié)構(gòu)破壞紊亂,脊髓伴隨脊膜向外膨出,多伴有炎性細胞侵襲。葉酸干預(yù)組中脊柱裂較少,無尾畸形較多,相比實驗組而言葉酸干預(yù)組發(fā)育有所改善,但比正常組發(fā)育仍然較差。2.各組神經(jīng)管中H3K27me3表達量的檢測:免疫組化結(jié)果顯示:正常組中H3K27me3的表達量低,實驗組中為強陽性表達,葉酸干預(yù)組中其表達量稍低于實驗組,但并沒有顯示出差異性,而與正常組相比較,差異較明顯,具有統(tǒng)計學(xué)意義(P0.05)。Western blot檢測結(jié)果顯示:實驗組中H3K27me3的表達量高于正常組和葉酸干預(yù)組,差異有統(tǒng)計學(xué)意義(P0.05),葉酸干預(yù)組表達量介于兩組之間,差異明顯,具有統(tǒng)計學(xué)意義(P0.05)。3.各組神經(jīng)管中Adcy2基因的QRT-PCR檢測結(jié)果顯示:在實驗組中高表達,與正常組和葉酸干預(yù)組比較,差異具有統(tǒng)計學(xué)意義(P0.05);在葉酸干預(yù)組中的表達量介于兩組之間,差異具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論1.葉酸干預(yù),可以有效降低神經(jīng)管畸形率的發(fā)生;2.神經(jīng)管畸形胎鼠中,H3K27me3表達水平上調(diào),葉酸代謝相關(guān)基因Adcy2的表達上調(diào),葉酸干預(yù)可以有效降低兩者的表達量。
[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.

【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R715.3
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本文編號：1491086