发布时间：2018-09-09 19:39
【摘要】： 背景: 肌-眼-脑病是一种常染色体隐性遗传性疾病,为编码N-乙酰氨基葡萄糖-氧位-甘露糖转移酶1(protein-O-linked mannoseβ1,2 -N-acetylglucosaminyl -transferase1,POMGnT1)的基因缺陷,临床表现为严重的肌营养不良、眼缺陷以及智力障碍。为了阐明该病的病理生理机制已经进行了许多研究,发现患者或者动物模型的脑内存在神经元迁移异常。然而,迄今为止,神经元迁移异常是如何发生的、生物体内还有哪些继发变化等问题还没有得到解决。 目的: 探讨肌-眼-脑病的病理生理机制,了解该病神经元迁移缺陷的发生过程以及基因表达谱的变化。方法: 1.产生POMGnT-1基因敲除小鼠并确定基因型,以同一窝的野生型小鼠作为正常对照。 2.应用免疫荧光染色的方法显示小脑基底膜层、胶质细胞界膜和颗粒细胞的发育;免疫荧光双标染色的方法显示Laminin和胶质细胞酸性蛋白之间的关系;应用高尔基染色显示小脑的细胞形态。 3.进行BrdU皮下注射,免疫标记识别S期的分裂细胞,进行出生后日期计数。 4.应用电镜分析来检查软脑膜基底膜层的完整性和颗粒细胞的分化情况。 5.应用YFPH小鼠显示由黄色荧光蛋白标记的苔藓纤维。 6.分离小脑皮质,提取mRNA。 7.利用Affymetrix公司的基因芯片,获取POMGnT-1基因敲除小鼠小脑皮质的基因差异表达谱,通过该公司的信息分析中心,进一步分析微阵列信息。 8.合成cDNA,行实时定量PCR证实微阵列数据。 结果: 1.POMGnT-1基因敲除小鼠的软脑膜基底膜层局灶的缺口和胶质细胞界膜的破坏与外生发层的细胞异位明确相关。Bergman胶质细胞的纤维回缩、紊乱、或突出到异位区域,缺乏正常的Bergman胶质细胞纤维的脚手架是导致神经元迁移失败的原因。不过,异位的EGL细胞可以发育成成熟的颗粒细胞,并与苔藓纤维形成突触。 2.和野生型相比较,POMGnT-1缺陷小鼠的大多数的基因的表达水平增加。在这些基因中,S100钙结合蛋白、酪氨酸羟化酶、小脑锌指样蛋白、calbindin 2、鸟苷酸结合蛋白增加的fold数较高,分别为27.39、13.97、13.94、11.64、11.00;仅有少数的几个基因的表达水平下降,分别为神经丝3-M、POMGnT-1和一个编码未知蛋白的基因。表达变化的基因分成转录、代谢、发育、信号转导、迁移、糖基化等组,其中,转录、代谢、发育、信号转导组发生变化的基因较多。 结论: 1.在POMGnT-1基因敲除小鼠中,导致小脑颗粒细胞异位的原因是软脑膜基底膜层和胶质细胞的界膜的破裂,而颗粒细胞迁移到IGL并非是分化所必需。 2.POMGnT-1的基因缺陷小鼠的基因表达谱则为进一步研究MEB的病理生理机制提供了新的线索。
[Abstract]:Background: myooculmonary encephalopathy is an autosomal recessive genetic disease. It is a gene defect encoding protein-O-linked mannose 尾 1- N-acetylglucosaminyl -transferase 1 (POMGnT1), which encodes N-acetylglucosaminyl -transferase1 (POMGnT1). Clinical manifestations include severe muscular dystrophy, eye defects, and mental retardation. In order to elucidate the pathophysiological mechanism of the disease, many studies have been carried out and abnormal neuronal migration has been found in the brain of patients or animal models. However, up to now, the problems of how the abnormal neuronal migration occurs and what secondary changes exist in the organism have not been solved. Objective: to investigate the pathophysiological mechanism of myo-eye-encephalopathy and to understand the process of neuronal migration defect and the change of gene expression profile. Methods: 1. POMGnT-1 gene knockout mice were produced and genotypes were determined, using the same litter of wild type mice as normal control. Immunofluorescence staining was used to show the development of cerebellar basement membrane, glial cell boundary membrane and granulosa cells, and immunofluorescence double labeling method was used to show the relationship between Laminin and glial acidic protein. Golgi staining was used to display the morphology of cerebellar cells. Subcutaneous injection of BrdU, immunolabeled identification of S phase mitotic cells, postnatal date count. 4. 4. The integrity of the basement membrane and the differentiation of granulosa cells were examined by electron microscopy. 5. 5. The moss fiber labeled by yellow fluorescent protein was shown in YFPH mice. 6. 6. The cerebellar cortex was isolated and mRNA. 7. The gene differential expression profiles of cerebellar cortex of POMGnT-1 knockout mice were obtained by using Affymetrix gene chip, and the microarray information was further analyzed by the company's information analysis center. Synthetic cDNA, was used to verify the microarray data by real time quantitative PCR. Results: 1.POMGnT-1 gene knockout mice showed a clear correlation between the gap in the basement membrane of pia and the destruction of the intercellular membrane in the ectopic of the ectopic cells of the ectopic hair layer. The fibers of the glial cells in the ectopic layer were retracted and disordered. The lack of normal Bergman glial fiber scaffold is the cause of neuronal migration failure. However, ectopic EGL cells can develop into mature granulosa cells and form synapses with moss fibers. 2. 2. The expression level of most genes in POMGnT-1 deficient mice was higher than that in wild type mice. In these genes, Ca-S100, tyrosine hydroxylase, cerebellar zinc finger like protein calbindin 2 and guanosine binding protein increased the number of fold, respectively, by 27.39% 13.9713.94.6411.00, and only a few genes decreased. They are neurofilament 3-MN POMGnT-1 and a gene encoding unknown protein respectively. The gene expression changes were divided into transcriptional, metabolic, developmental, signal transduction, migration, glycosylation and other groups, among which, transcription, metabolism, development, signal transduction group changed more genes. Conclusion: 1. In POMGnT-1 knockout mice, ectopia of cerebellar granulosa cells was caused by the rupture of the basement membrane of the pia mater and the boundary membrane of the glial cells. However, the migration of granulosa cells to IGL is not necessary for differentiation. The gene expression profiles of 2.POMGnT-1 gene deficient mice provide a new clue for further study of pathophysiological mechanism of MEB.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363
本文编号：2233375
[Abstract]:Background: myooculmonary encephalopathy is an autosomal recessive genetic disease. It is a gene defect encoding protein-O-linked mannose 尾 1- N-acetylglucosaminyl -transferase 1 (POMGnT1), which encodes N-acetylglucosaminyl -transferase1 (POMGnT1). Clinical manifestations include severe muscular dystrophy, eye defects, and mental retardation. In order to elucidate the pathophysiological mechanism of the disease, many studies have been carried out and abnormal neuronal migration has been found in the brain of patients or animal models. However, up to now, the problems of how the abnormal neuronal migration occurs and what secondary changes exist in the organism have not been solved. Objective: to investigate the pathophysiological mechanism of myo-eye-encephalopathy and to understand the process of neuronal migration defect and the change of gene expression profile. Methods: 1. POMGnT-1 gene knockout mice were produced and genotypes were determined, using the same litter of wild type mice as normal control. Immunofluorescence staining was used to show the development of cerebellar basement membrane, glial cell boundary membrane and granulosa cells, and immunofluorescence double labeling method was used to show the relationship between Laminin and glial acidic protein. Golgi staining was used to display the morphology of cerebellar cells. Subcutaneous injection of BrdU, immunolabeled identification of S phase mitotic cells, postnatal date count. 4. 4. The integrity of the basement membrane and the differentiation of granulosa cells were examined by electron microscopy. 5. 5. The moss fiber labeled by yellow fluorescent protein was shown in YFPH mice. 6. 6. The cerebellar cortex was isolated and mRNA. 7. The gene differential expression profiles of cerebellar cortex of POMGnT-1 knockout mice were obtained by using Affymetrix gene chip, and the microarray information was further analyzed by the company's information analysis center. Synthetic cDNA, was used to verify the microarray data by real time quantitative PCR. Results: 1.POMGnT-1 gene knockout mice showed a clear correlation between the gap in the basement membrane of pia and the destruction of the intercellular membrane in the ectopic of the ectopic cells of the ectopic hair layer. The fibers of the glial cells in the ectopic layer were retracted and disordered. The lack of normal Bergman glial fiber scaffold is the cause of neuronal migration failure. However, ectopic EGL cells can develop into mature granulosa cells and form synapses with moss fibers. 2. 2. The expression level of most genes in POMGnT-1 deficient mice was higher than that in wild type mice. In these genes, Ca-S100, tyrosine hydroxylase, cerebellar zinc finger like protein calbindin 2 and guanosine binding protein increased the number of fold, respectively, by 27.39% 13.9713.94.6411.00, and only a few genes decreased. They are neurofilament 3-MN POMGnT-1 and a gene encoding unknown protein respectively. The gene expression changes were divided into transcriptional, metabolic, developmental, signal transduction, migration, glycosylation and other groups, among which, transcription, metabolism, development, signal transduction group changed more genes. Conclusion: 1. In POMGnT-1 knockout mice, ectopia of cerebellar granulosa cells was caused by the rupture of the basement membrane of the pia mater and the boundary membrane of the glial cells. However, the migration of granulosa cells to IGL is not necessary for differentiation. The gene expression profiles of 2.POMGnT-1 gene deficient mice provide a new clue for further study of pathophysiological mechanism of MEB.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363
【参考文献】
相关期刊论文 前2条
1 郝虎;宋元宗;肖昕;张日佳;刘振寰;;双胞胎同患肌-眼-脑病[J];中国当代儿科杂志;2006年05期
2 容敏华;何敏;黄千贻;;运用Affymetrix网上分析中心初步分析EGCG作用肝癌细胞SMMC-7721前后差异表达基因数据[J];医学信息;2007年01期
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