肌醇代谢障碍在神经管畸形发生中的作用机制研究

发布时间：2017-12-27 22:22

本文关键词：肌醇代谢障碍在神经管畸形发生中的作用机制研究　出处：《北京工业大学》2015年硕士论文　论文类型：学位论文

【摘要】：神经管畸形(Neural tube defects,NTDs)是最常见、最严重的出生缺陷之一,发病率高,临床后果严重,也是中国儿童出生缺陷的重要问题。目前,该疾病的发生机制尚未阐明,是出生缺陷领域研究的重点难点之一。除叶酸外,肌醇及其代谢通路对NTDs的影响也是尚待阐明的病因学问题之一。肌醇(Inositol)属于维生素B族物质,是一种细胞生长与存活的重要营养物。研究表明,肌醇与NTDs发生密切相关,肌醇及其代谢通路在胚胎发育尤其是在神经管发育过程中发挥着重要作用,但其确切机制尚不明确。本研究从肌醇营养物代谢的角度出发,应用已建立的人类NTD出生缺陷临床样本资源库,并干预肌醇代谢过程中重要靶点酶,建立肌醇代谢障碍的NTD小鼠动物模型;采用气相色谱质谱联用技术(GC-MS)、免疫组化、RT-PCR及Western Blotting等技术和方法,进一步研究肌醇代谢障碍在NTDs发生过程中的作用机制,结果如下:1、从已建立的人类NTD样本库中,随机抽取NTD及正常对照孕妇血清样本各40例和胎儿神经组织样本各9例,采用GC-MS与Western Blotting方法分别对人类NTD孕妇血清肌醇水平及胎儿神经组织PI3K/AKT信号通路活性进行检测,发现NTD孕妇血清中肌醇水平显著低于正常对照组(1.8676±0.7270 mg/L2.5779±1.0951 mg/L,P0.05);NTD胎儿神经组织中p AKT-S473磷酸化蛋白表达明显高于正常对照组(P0.05),AKT磷酸化水平提高,PI3K/AKT信号通路过度激活。2、采用Li2CO3特异性抑制C57BL/6J小鼠体内肌醇合成代谢靶点酶(IMPase)活性,成功建立了肌醇代谢障碍NTD小鼠动物模型,发现孕第7.5天腹腔注射350mg/kg剂量Li2CO3为最佳造模方案,NTDs发生率高达26.7%,其主要表型脑膨出和无脑畸形。孕鼠血清中肌醇水平在350 mg/kg Li2CO3剂量注射后4~8 h期间处于最低(P0.01),16 h后仍低于正常水平(P0.05)。HE染色发现,脑膨出小鼠胚胎神经组织和细胞形态发生变化,端脑神经管闭合失败程度较后脑更严重。3、在已建立的肌醇代谢障碍NTD小鼠动物模型基础上,采用免疫组化、RT-PCR及Western Blotting等方法检测NTD小鼠胚胎神经组织中PI3K/AKT信号通路活性及相关基因(Pten、Pik3ca、Akt1、Inpp5e)的转录水平,进一步探讨肌醇缺乏对该通路的调控作用。结果发现,p AKT-S473磷酸化蛋白主要定位于神经上皮细胞室管膜层结构,且在NTD胚胎神经组织的表达明显高于正常对照组(P0.05),AKT磷酸化水平升高,PI3K/AKT信号通路过度激活;与正常对照组相比,Pik3ca基因转录水平明显升高(P0.05),Pten基因转录水平显著降低(P0.05),而Inpp5e和Akt1基因转录水平无明显变化。本研究结果表明,母体肌醇代谢障碍和NTDs发生密切相关。实验成功建立了肌醇代谢障碍NTD小鼠动物模型,发现肌醇缺乏可能主要通过Pik3ca和Pten基因的协同作用而过度激活端脑神经上皮PI3K/AKT信号通路,影响神经组织结构和细胞形态,干扰胎儿神经管Closer 2闭合过程从而导致NTDs发生。因此,本研究结果将对肌醇在NTDs发生中作用机制的进一步阐明及NTDs早期诊断和预防具有重要意义。
[Abstract]:Neural tube defects (NTDs) is one of the most common and serious birth defects, with high incidence and serious clinical consequences. It is also an important problem of birth defects in China. At present, the mechanism of the disease has not been elucidated, which is one of the key difficulties in the field of birth defect research. In addition to folic acid, the effect of inositol and its metabolic pathway on NTDs is also one of the aetiology problems that have yet to be elucidated. Inositol (Inositol) belongs to the vitamin B group and is an important nutrient for cell growth and survival. Studies have shown that inositol is closely related to NTDs. Inositol and its metabolic pathway play an important role in embryonic development, especially in the development of neural tube, but the exact mechanism is not clear. This study from the perspective of inositol nutrient metabolism, application of established human birth defects of NTD clinical sample resource library, and intervention of important target enzyme inositol metabolism, NTD mice to establish animal models of myo inositol metabolism; by using gas chromatography-mass spectrometry (GC-MS), immunohistochemistry, RT-PCR and Western Blotting technology and method of further research in the mechanism of inositol metabolism, in the process of NTDs, the results are as follows: 1 samples from human NTD library has been established, randomly selected NTD and normal pregnant women serum samples of 40 cases and 9 cases of fetal neural tissue samples, using GC-MS and Western methods respectively to Blotting the activity of PI3K/AKT pathway in human NTD serum levels of inositol in pregnant women and fetal neural tissue were detected and found the levels of inositol NTD in serum of pregnant women was significantly lower than the normal control group (1.8676 + 0.7270 mg/L2. 5779 + 1.0951 mg/L, P0.05); the expression of P AKT-S473 phosphorylated protein in NTD fetal nerve tissue was significantly higher than that in the normal control group (P0.05), the level of AKT phosphorylation was increased, and PI3K/AKT signaling pathway was overactivated. 2, using Li2CO3 specific inhibitor of inositol metabolism in C57BL/6J mice target enzyme (IMPase) activity, successfully established NTD animal model of phosphoinositide metabolism disorder, found pregnant the first 7.5 days of intraperitoneal injection of 350mg/kg dose of Li2CO3 was the best modeling scheme, the incidence rate of NTDs up to 26.7%, the main phenotype and anencephaly encephalocele. The level of inositol in serum of pregnant rats was lowest (P0.01) at 4~8 h after injection of 350 mg/kg Li2CO3 (P0.01), and still lower than that of normal level after 16 h (P0.05). HE staining showed that the neural tissue and cell morphology of the encephalocele mouse embryos were changed, and the failure degree of the closure of the end of the brain was more serious than that of the posterior brain. 3, based on inositol metabolic disorders in NTD mice animal model has been established, the activity of PI3K/AKT signal pathway and related genes by immunohistochemistry, RT-PCR Western and Blotting NTD method for the detection of mouse embryonic neural tissue (Pten, Pik3ca, Akt1, Inpp5e) transcription level, to further explore the lack of regulation of the inositol pathway. The results showed that P phosphorylation of AKT-S473 protein was mainly localized in neural tube epithelial cell room film structure, and the expression of NTD in embryonic neural tissue is significantly higher than the normal control group (P0.05), increased the phosphorylation level of AKT, PI3K/AKT signaling pathway activated; compared with normal control group, the transcription level of Pik3ca gene increased significantly (P0.05). The transcription level of Pten gene decreased significantly (P0.05), and Inpp5e and Akt1 gene transcription level did not change significantly. The results of this study suggest that the maternal inositol metabolic disorder is closely related to the occurrence of NTDs. The experiment successfully established the animal model of mice NTD inositol metabolism, found that inositol deficiency may mainly through the synergistic effect of Pik3ca and Pten gene and cerebral neuroepithelial excessive activation of PI3K/AKT signaling pathway, effect of nerve tissue structure and cell morphology, interference of fetal neural tube closure process leading to Closer 2 NTDs. Therefore, the results of this study will be of great significance to the further clarification of the mechanism of inositol in the occurrence of NTDs and the early diagnosis and prevention of NTDs.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R714.5

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