發(fā)布時間：2018-09-09 19:39
【摘要】： 背景: 肌-眼-腦病是一種常染色體隱性遺傳性疾病,為編碼N-乙酰氨基葡萄糖-氧位-甘露糖轉(zhuǎn)移酶1(protein-O-linked mannoseβ1,2 -N-acetylglucosaminyl -transferase1,POMGnT1)的基因缺陷,臨床表現(xiàn)為嚴(yán)重的肌營養(yǎng)不良、眼缺陷以及智力障礙。為了闡明該病的病理生理機制已經(jīng)進行了許多研究,發(fā)現(xiàn)患者或者動物模型的腦內(nèi)存在神經(jīng)元遷移異常。然而,迄今為止,神經(jīng)元遷移異常是如何發(fā)生的、生物體內(nèi)還有哪些繼發(fā)變化等問題還沒有得到解決。 目的: 探討肌-眼-腦病的病理生理機制,了解該病神經(jīng)元遷移缺陷的發(fā)生過程以及基因表達譜的變化。方法: 1.產(chǎn)生POMGnT-1基因敲除小鼠并確定基因型,以同一窩的野生型小鼠作為正常對照。 2.應(yīng)用免疫熒光染色的方法顯示小腦基底膜層、膠質(zhì)細胞界膜和顆粒細胞的發(fā)育;免疫熒光雙標(biāo)染色的方法顯示Laminin和膠質(zhì)細胞酸性蛋白之間的關(guān)系;應(yīng)用高爾基染色顯示小腦的細胞形態(tài)。 3.進行BrdU皮下注射,免疫標(biāo)記識別S期的分裂細胞,進行出生后日期計數(shù)。 4.應(yīng)用電鏡分析來檢查軟腦膜基底膜層的完整性和顆粒細胞的分化情況。 5.應(yīng)用YFPH小鼠顯示由黃色熒光蛋白標(biāo)記的苔蘚纖維。 6.分離小腦皮質(zhì),提取mRNA。 7.利用Affymetrix公司的基因芯片,獲取POMGnT-1基因敲除小鼠小腦皮質(zhì)的基因差異表達譜,通過該公司的信息分析中心,進一步分析微陣列信息。 8.合成cDNA,行實時定量PCR證實微陣列數(shù)據(jù)。 結(jié)果: 1.POMGnT-1基因敲除小鼠的軟腦膜基底膜層局灶的缺口和膠質(zhì)細胞界膜的破壞與外生發(fā)層的細胞異位明確相關(guān)。Bergman膠質(zhì)細胞的纖維回縮、紊亂、或突出到異位區(qū)域,缺乏正常的Bergman膠質(zhì)細胞纖維的腳手架是導(dǎo)致神經(jīng)元遷移失敗的原因。不過,異位的EGL細胞可以發(fā)育成成熟的顆粒細胞,并與苔蘚纖維形成突觸。 2.和野生型相比較,POMGnT-1缺陷小鼠的大多數(shù)的基因的表達水平增加。在這些基因中,S100鈣結(jié)合蛋白、酪氨酸羥化酶、小腦鋅指樣蛋白、calbindin 2、鳥苷酸結(jié)合蛋白增加的fold數(shù)較高,分別為27.39、13.97、13.94、11.64、11.00;僅有少數(shù)的幾個基因的表達水平下降,分別為神經(jīng)絲3-M、POMGnT-1和一個編碼未知蛋白的基因。表達變化的基因分成轉(zhuǎn)錄、代謝、發(fā)育、信號轉(zhuǎn)導(dǎo)、遷移、糖基化等組,其中,轉(zhuǎn)錄、代謝、發(fā)育、信號轉(zhuǎn)導(dǎo)組發(fā)生變化的基因較多。 結(jié)論: 1.在POMGnT-1基因敲除小鼠中,導(dǎo)致小腦顆粒細胞異位的原因是軟腦膜基底膜層和膠質(zhì)細胞的界膜的破裂,而顆粒細胞遷移到IGL并非是分化所必需。 2.POMGnT-1的基因缺陷小鼠的基因表達譜則為進一步研究MEB的病理生理機制提供了新的線索。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R363
本文編號：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R363
【參考文獻】
相關(guān)期刊論文 前2條
1 郝虎;宋元宗;肖昕;張日佳;劉振寰;;雙胞胎同患肌-眼-腦病[J];中國當(dāng)代兒科雜志;2006年05期
2 容敏華;何敏;黃千貽;;運用Affymetrix網(wǎng)上分析中心初步分析EGCG作用肝癌細胞SMMC-7721前后差異表達基因數(shù)據(jù)[J];醫(yī)學(xué)信息;2007年01期
，本文編號：2233375
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