C1型尼曼—匹克病小鼠神經(jīng)系統(tǒng)中髓鞘形成障礙及microRNA差異性表達研究
發(fā)布時間：2018-11-04 15:29
【摘要】：背景C1型尼曼-匹克病(NPC1)是由于Npc1基因突變引起的一種神經(jīng)退行性疾病,目前尚無有效的治愈方法。研究發(fā)現(xiàn),NPC1患者腦中存在嚴重的髓鞘形成障礙,如果不能及時和有效的治療,將會影響神經(jīng)的正常傳導,導致神經(jīng)軸突的變性以及神經(jīng)元的死亡,引發(fā)嚴重的神經(jīng)功能損傷。microRNA(miRNA)及其靶基因構(gòu)建了細胞內(nèi)全方位多層次的基因表達調(diào)控網(wǎng)絡,在髓鞘形成及損傷中扮演關鍵的角色,參與調(diào)節(jié)神經(jīng)系統(tǒng)的發(fā)育過程及神經(jīng)退行性疾病的發(fā)生。因此,我們從髓鞘形成障礙入手,初步研究與髓鞘形成密切相關的miR-205-5p(miR-205)及其靶基因,并闡釋其分子作用機制,研究Npc1-/-小鼠髓鞘形成過程,為治療脫髓鞘性疾病提供新的基因及藥物靶點,最終為臨床的早期診斷及預防提供重要的參考依據(jù)。目的1.研究Npc1-/-小鼠不同發(fā)育時期腦組織不同部位的髓鞘堿性蛋白(myelin basic protein,MBP)的表達和分布情況,揭示Npc1-/-小鼠腦組織髓鞘形成的表達模式;2.通過高通量Small RNA測序技術,篩選Npc1-/-小鼠腦中有顯著差異的mi RNAs;3.對Npc1-/-小鼠腦中差異表達的miRNAs,進行靶基因預測及IPA功能與疾病分析,初步探討miRNA參與NPC1髓鞘形成調(diào)控的分子機制。方法1.在小鼠出生后第7天(postnatal day 7,P7)進行基因型鑒定,Npc1-/-和Npc1+/+小鼠將被用于下列實驗;2.分別取P9,P35,P60天小鼠,進行心臟灌注。分離完整腦組織進行4%PFA固定,18%蔗糖溶液脫水。OCT包埋劑包埋冷凍切片后,經(jīng)免疫熒光組織化學方法分析小鼠腦組織在不同時期不同部位MBP蛋白表達情況;3.Western blotting技術檢測P9,P35,P60天小鼠腦組織不同部位MBP蛋白表達情況,并用Fiji/ImageJ進行灰度掃描,定量分析。qPCR檢測P35 Npc1-/-和Npc1+/+小鼠大腦皮層和海馬組織中髓鞘形成相關蛋白表達情況;4.取P35天Npc1-/-和Npc1+/+小鼠海馬組織進行Small RNA測序分析,篩選差異表達的miRNAs;5.對Npc1-/-小鼠中差異表達的mi RNAs進行靶基因預測及IPA功能與疾病分析;6.選擇差異表達最顯著的miR-205,通過qPCR檢測Npc1-/-小鼠海馬中miR-205及其靶基因的表達情況。結(jié)果1.隨著小鼠的生長發(fā)育,腦中MBP蛋白的表達呈逐漸增加趨勢。而Npc1-/-小鼠腦組織各部位MBP蛋白的表達均顯著下降,且不同部位MBP呈現(xiàn)不同的表達模式;2.qPCR檢測P35天Npc1-/-和Npc1+/+小鼠大腦皮層和海馬組織中PLP,MAG,MOG和CNPase的表達情況,發(fā)現(xiàn)表達量均呈顯著性降低;3.通過Small RNA測序分析,發(fā)現(xiàn)Npc1-/-小鼠海馬中差異表達的miRNAs共有59個(P0.05),其中表達上調(diào)27個,表達下調(diào)32個;4.對差異表達的miRNAs進行靶基因預測以及IPA疾病與功能分析,發(fā)現(xiàn)髓鞘形成障礙和脫髓鞘被顯著富集,表明miRNA參與了NPC1髓鞘形成的調(diào)控;5.qPCR分析表明Npc1-/-小鼠海馬中miR-205表達顯著升高,其靶基因的表達呈現(xiàn)不同程度的降低,且Npc1-/-小鼠海馬中MBP,PLP,MAG,MOG和CNPase蛋白表達顯著降低,初步表明在Npc1-/-小鼠海馬中miR-205通過其靶基因參與髓鞘形成的調(diào)控。結(jié)論1.Npc1-/-小鼠腦中髓鞘形成主要標志物MBP蛋白的表達模式具有時空差異性,且MBP,PLP等蛋白的表達顯著下調(diào),揭示Npc1基因的突變導致髓鞘形成障礙;2.Npc1-/-小鼠腦中差異表達的mi RNA參與了NPC1疾病的發(fā)生發(fā)展;3.Npc1-/-小鼠海馬中miRNA-205的表達上調(diào)導致其靶基因表達下調(diào),且髓鞘標志物MBP,PLP等蛋白表達顯著下調(diào),初步揭示miRNA-205在Npc1-/-小鼠海馬中通過靶基因Csf1、Abcd1、Fam126a和Lpar1調(diào)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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R596
本文編號：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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R596
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