C1型尼曼—匹克病小鼠神经系统中髓鞘形成障碍及microRNA差异性表达研究
发布时间：2018-11-04 15:29
【摘要】：背景C1型尼曼-匹克病(NPC1)是由于Npc1基因突变引起的一种神经退行性疾病,目前尚无有效的治愈方法。研究发现,NPC1患者脑中存在严重的髓鞘形成障碍,如果不能及时和有效的治疗,将会影响神经的正常传导,导致神经轴突的变性以及神经元的死亡,引发严重的神经功能损伤。microRNA(miRNA)及其靶基因构建了细胞内全方位多层次的基因表达调控网络,在髓鞘形成及损伤中扮演关键的角色,参与调节神经系统的发育过程及神经退行性疾病的发生。因此,我们从髓鞘形成障碍入手,初步研究与髓鞘形成密切相关的miR-205-5p(miR-205)及其靶基因,并阐释其分子作用机制,研究Npc1-/-小鼠髓鞘形成过程,为治疗脱髓鞘性疾病提供新的基因及药物靶点,最终为临床的早期诊断及预防提供重要的参考依据。目的1.研究Npc1-/-小鼠不同发育时期脑组织不同部位的髓鞘碱性蛋白(myelin basic protein,MBP)的表达和分布情况,揭示Npc1-/-小鼠脑组织髓鞘形成的表达模式;2.通过高通量Small RNA测序技术,筛选Npc1-/-小鼠脑中有显著差异的mi RNAs;3.对Npc1-/-小鼠脑中差异表达的miRNAs,进行靶基因预测及IPA功能与疾病分析,初步探讨miRNA参与NPC1髓鞘形成调控的分子机制。方法1.在小鼠出生后第7天(postnatal day 7,P7)进行基因型鉴定,Npc1-/-和Npc1+/+小鼠将被用于下列实验;2.分别取P9,P35,P60天小鼠,进行心脏灌注。分离完整脑组织进行4%PFA固定,18%蔗糖溶液脱水。OCT包埋剂包埋冷冻切片后,经免疫荧光组织化学方法分析小鼠脑组织在不同时期不同部位MBP蛋白表达情况;3.Western blotting技术检测P9,P35,P60天小鼠脑组织不同部位MBP蛋白表达情况,并用Fiji/ImageJ进行灰度扫描,定量分析。qPCR检测P35 Npc1-/-和Npc1+/+小鼠大脑皮层和海马组织中髓鞘形成相关蛋白表达情况;4.取P35天Npc1-/-和Npc1+/+小鼠海马组织进行Small RNA测序分析,筛选差异表达的miRNAs;5.对Npc1-/-小鼠中差异表达的mi RNAs进行靶基因预测及IPA功能与疾病分析;6.选择差异表达最显著的miR-205,通过qPCR检测Npc1-/-小鼠海马中miR-205及其靶基因的表达情况。结果1.随着小鼠的生长发育,脑中MBP蛋白的表达呈逐渐增加趋势。而Npc1-/-小鼠脑组织各部位MBP蛋白的表达均显著下降,且不同部位MBP呈现不同的表达模式;2.qPCR检测P35天Npc1-/-和Npc1+/+小鼠大脑皮层和海马组织中PLP,MAG,MOG和CNPase的表达情况,发现表达量均呈显著性降低;3.通过Small RNA测序分析,发现Npc1-/-小鼠海马中差异表达的miRNAs共有59个(P0.05),其中表达上调27个,表达下调32个;4.对差异表达的miRNAs进行靶基因预测以及IPA疾病与功能分析,发现髓鞘形成障碍和脱髓鞘被显著富集,表明miRNA参与了NPC1髓鞘形成的调控;5.qPCR分析表明Npc1-/-小鼠海马中miR-205表达显著升高,其靶基因的表达呈现不同程度的降低,且Npc1-/-小鼠海马中MBP,PLP,MAG,MOG和CNPase蛋白表达显著降低,初步表明在Npc1-/-小鼠海马中miR-205通过其靶基因参与髓鞘形成的调控。结论1.Npc1-/-小鼠脑中髓鞘形成主要标志物MBP蛋白的表达模式具有时空差异性,且MBP,PLP等蛋白的表达显著下调,揭示Npc1基因的突变导致髓鞘形成障碍;2.Npc1-/-小鼠脑中差异表达的mi RNA参与了NPC1疾病的发生发展;3.Npc1-/-小鼠海马中miRNA-205的表达上调导致其靶基因表达下调,且髓鞘标志物MBP,PLP等蛋白表达显著下调,初步揭示miRNA-205在Npc1-/-小鼠海马中通过靶基因Csf1、Abcd1、Fam126a和Lpar1调控髓鞘形成。
[Abstract]:Background C1-type NPC1 is a neurodegenerative disease caused by the mutation of Npc1 gene mutation, and there is no effective cure method at present. The study found that there was a severe neurological disorder in the brain of NPC1, which could affect the normal conduction of nerves, resulting in degeneration of nerve axons and death of neurons, causing serious neurological damage. MicroRNA (miRNA) and its target gene construct all-round multi-level gene expression regulatory network in the cell, play a key role in the formation and damage of nerve cells, and participate in regulating the development of nervous system and the occurrence of neurodegenerative diseases. Therefore, we start with the formation of obstacles, and initially study the miR-205-5p (miR-205) and its target gene which are closely related to the formation of human body, and explain the mechanism of molecular action of the miR-205-5p (miR-205), and to study the formation process of Npc1-/-mice and provide new genes and drug targets for the treatment of inflammatory diseases. and finally provides an important reference basis for early diagnosis and prevention of clinic. Purpose 1. To investigate the expression and distribution of myelin basic protein (NPY) in different parts of Npc1-/-mice during different developmental stages, and to reveal the expression pattern of Npc1-/-mouse brain tissue formation. Through high-throughput Small RNA sequencing technology, there were significantly different mi-RNAs in Npc 1-/-mouse brain; 3. Using miRNAs differentially expressed in the brain of Npc 1-/-mice, target gene prediction and IPA function and disease analysis were carried out, and the molecular mechanism of miRNA involvement in the regulation of NPC1-/-mouse brain was discussed. Method 1. Genotyping, Npc1-/-and Npc1 +/ + mice will be used for the following experiments on day 7 (postnatal day 7, P7) after birth in mice; 2. The hearts were perfused with p35, P35 and P60 mice respectively. The complete brain tissue was isolated for 4% PFA fixation and 18% sucrose solution was dehydrated. After the frozen sections were embedded in the OCT package, the expression of p27 protein in different parts of the brain tissue of mice was analyzed by immunofluorescence histochemical method; 3. Western blotting technique was used to detect the expression of tau protein in different parts of brain tissues of mice brain tissues of mice, P35 and P60 days, and gray-scale scanning was carried out with Fiji/ ImageJ. Quantitative analysis. qPCR detection of P35 Npc1-/-and Npc1 +/ + mouse cerebral cortex and hippocampus resulted in the formation of related protein expression; 4. Small RNA sequencing was performed in the hippocampal tissue of Npc1-/-and Npc1 +/ + mice for P35 days to screen differentially expressed miRNAs; 5. The target gene prediction and the IPA function and disease analysis were carried out on the mi-RNAs differentially expressed in Npc 1-/-mice. The expression of miR-205 and its target gene in hippocampus of Npc1-/-mice was detected by qPCR. Result 1. With the development of mice, the expression of p27 protein in brain gradually increased. and the expression of PLP, MAG, MOG and CNPase in cortex and hippocampus of Npc1-/-and Npc1 +/ + mice were detected by qPCR. It was found that the expression level decreased significantly; 3. According to the analysis of Small RNA sequencing, 59 of the miRNAs differentially expressed in the hippocampus of Npc1-/-mice were found (P <0.05), of which 27 were up-regulated and 32 were down-regulated. The results showed that the expression of miR-205 in hippocampus of Npc1-/-mice was significantly higher than that of NPC1-/-mouse hippocampus. The expression of the target gene of Npc1-/-mouse hippocampus decreased significantly, and the expression of PLP, MAG, MOG and CNPase protein in the hippocampus of Npc1-/-mice was significantly decreased, and the expression of miR-205 in the hippocampus of Npc1-/-mice was preliminarily studied. Conclusion 1. The expression pattern of Npc1-/-mouse brain which forms the main marker is space-time difference, and the expression of p27, PLP and other proteins is down-regulated, revealing that the mutation of Npc1 gene leads to the formation of obstacles. 2. The expression of mi RNA differentially expressed in the brain of Npc1-/-mice was involved in the development of NPC1 disease; 3. The expression of miRNA-205 in hippocampus of Npc1-/-mouse resulted in down-regulation of target gene expression, and the expression of CD44v6, PLP and other proteins was down-regulated. The results showed that miRNA-205 was formed by target gene Csf1, Abcd1, Fam126a and Lpar1 in the hippocampus of Npc1-/-mice.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596
本文编号：2310261
[Abstract]:Background C1-type NPC1 is a neurodegenerative disease caused by the mutation of Npc1 gene mutation, and there is no effective cure method at present. The study found that there was a severe neurological disorder in the brain of NPC1, which could affect the normal conduction of nerves, resulting in degeneration of nerve axons and death of neurons, causing serious neurological damage. MicroRNA (miRNA) and its target gene construct all-round multi-level gene expression regulatory network in the cell, play a key role in the formation and damage of nerve cells, and participate in regulating the development of nervous system and the occurrence of neurodegenerative diseases. Therefore, we start with the formation of obstacles, and initially study the miR-205-5p (miR-205) and its target gene which are closely related to the formation of human body, and explain the mechanism of molecular action of the miR-205-5p (miR-205), and to study the formation process of Npc1-/-mice and provide new genes and drug targets for the treatment of inflammatory diseases. and finally provides an important reference basis for early diagnosis and prevention of clinic. Purpose 1. To investigate the expression and distribution of myelin basic protein (NPY) in different parts of Npc1-/-mice during different developmental stages, and to reveal the expression pattern of Npc1-/-mouse brain tissue formation. Through high-throughput Small RNA sequencing technology, there were significantly different mi-RNAs in Npc 1-/-mouse brain; 3. Using miRNAs differentially expressed in the brain of Npc 1-/-mice, target gene prediction and IPA function and disease analysis were carried out, and the molecular mechanism of miRNA involvement in the regulation of NPC1-/-mouse brain was discussed. Method 1. Genotyping, Npc1-/-and Npc1 +/ + mice will be used for the following experiments on day 7 (postnatal day 7, P7) after birth in mice; 2. The hearts were perfused with p35, P35 and P60 mice respectively. The complete brain tissue was isolated for 4% PFA fixation and 18% sucrose solution was dehydrated. After the frozen sections were embedded in the OCT package, the expression of p27 protein in different parts of the brain tissue of mice was analyzed by immunofluorescence histochemical method; 3. Western blotting technique was used to detect the expression of tau protein in different parts of brain tissues of mice brain tissues of mice, P35 and P60 days, and gray-scale scanning was carried out with Fiji/ ImageJ. Quantitative analysis. qPCR detection of P35 Npc1-/-and Npc1 +/ + mouse cerebral cortex and hippocampus resulted in the formation of related protein expression; 4. Small RNA sequencing was performed in the hippocampal tissue of Npc1-/-and Npc1 +/ + mice for P35 days to screen differentially expressed miRNAs; 5. The target gene prediction and the IPA function and disease analysis were carried out on the mi-RNAs differentially expressed in Npc 1-/-mice. The expression of miR-205 and its target gene in hippocampus of Npc1-/-mice was detected by qPCR. Result 1. With the development of mice, the expression of p27 protein in brain gradually increased. and the expression of PLP, MAG, MOG and CNPase in cortex and hippocampus of Npc1-/-and Npc1 +/ + mice were detected by qPCR. It was found that the expression level decreased significantly; 3. According to the analysis of Small RNA sequencing, 59 of the miRNAs differentially expressed in the hippocampus of Npc1-/-mice were found (P <0.05), of which 27 were up-regulated and 32 were down-regulated. The results showed that the expression of miR-205 in hippocampus of Npc1-/-mice was significantly higher than that of NPC1-/-mouse hippocampus. The expression of the target gene of Npc1-/-mouse hippocampus decreased significantly, and the expression of PLP, MAG, MOG and CNPase protein in the hippocampus of Npc1-/-mice was significantly decreased, and the expression of miR-205 in the hippocampus of Npc1-/-mice was preliminarily studied. Conclusion 1. The expression pattern of Npc1-/-mouse brain which forms the main marker is space-time difference, and the expression of p27, PLP and other proteins is down-regulated, revealing that the mutation of Npc1 gene leads to the formation of obstacles. 2. The expression of mi RNA differentially expressed in the brain of Npc1-/-mice was involved in the development of NPC1 disease; 3. The expression of miRNA-205 in hippocampus of Npc1-/-mouse resulted in down-regulation of target gene expression, and the expression of CD44v6, PLP and other proteins was down-regulated. The results showed that miRNA-205 was formed by target gene Csf1, Abcd1, Fam126a and Lpar1 in the hippocampus of Npc1-/-mice.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596
【参考文献】
相关期刊论文 前4条
1 闫欣;乔梁;杨恩慧;林俊堂;;C1型尼曼-匹克氏症小鼠嗅球胶质细胞的活性变化[J];生理学报;2016年02期
2 杨光;李翠;杨文静;张溢凡;肖林;;MicroRNA调控少突胶质细胞分化的研究进展[J];生命科学;2013年03期
3 陆沭华;张彦明;孙雨梅;赵广圣;刘立明;张兴霞;何广胜;;尼曼-匹克病合并骨髓转移癌1例并文献复习[J];苏州大学学报(医学版);2010年05期
4 孙瑞红,林世和,张艳;tau蛋白和中枢神经系统变性疾病[J];中华神经科杂志;2004年01期
，本文编号：2310261
本文链接：https://www.wllwen.com/yixuelunwen/nfm/2310261.html
最近更新
教材专著
