miR-26a对小鼠神经元形态发育影响的研究

发布时间：2019-02-27 10:25

【摘要】：目的：探讨microRNA-26a (miR-26a)在神经元形态发育过程中发挥的作用及其分子机制研究。方法：1.运用实时荧光定量PCR的方法，检测miR-26a在神经干细胞和神经元细胞中的表达情况。2.用免疫荧光染色和激光共聚焦的方法检测收集神经元细胞图像。3.利用Image-ProPlus软件对神经元细胞形态进行Sholl分析并统计神经元突起的长度和分布。4.利用Targetscan软件对miR-26a进行靶点预测分析。5.在神经元细胞中通过转染miR-26a mimic实现miR-26a的特异性过表达；转染miR-26ainhibitor实现miR-26a的特异性抑制。通过慢病毒感染法实现phosphatase and tensin homolog (PTEN)的特异性过表达；利用PTENshRNA通过small interfering RNA(siRNA)技术实现对PTEN的特异性敲降。并通过蛋白质印迹反应检测PTEN蛋白的表达水平。结果：1.miR-26a在神经元细胞中的表达比在神经干细胞中的表达量要丰富。2.过表达miR-26a组神经元细胞突起的数量、长度和分布较对照组有着明显升高；敲降miR-26a组神经元细胞的突起的数量、长度和分布较对照组有着明显下降（n100, P0.05）。3.PTEN信使RNA（mRNA）的3’端非编译区(3’UTR)含有三个与miR-26a特异性结合的位点。4.在神经元细胞中，过表达miR-26a，PTEN蛋白表达水平出现明显下降。过表达PTEN，神经元细胞突起的数量、长度和分布均出现显著性下降；而敲降PTEN，神经元细胞突起的数量、长度和分布均出现显著性上升（n100, P0.05）。结论：1.过表达miR-26a能够促进神经元细胞突起的生长，，增加神经元细胞突起的数量、长度和分布，表明miR-26a在神经元形态发育过程中发挥着重要的作用。2.在神经元细胞中PTEN是miR-26a的一个关键性作用靶点。3.过表达PTEN基因能够明显抑制神经元细胞的生长发育，降低神经元细胞的可塑性。在神经元细胞中PTEN是介导miR-26a发挥其相关调控作用的关键分子。这些发现让人们对神经元可塑性及其生长有了更进一步的认识，能够为阿尔茨海默症及其相关神经退行性疾病提供一个新的解决思路。
[Abstract]:Aim: to investigate the role of microRNA-26a (miR-26a) in neuronal morphological development and its molecular mechanism. Methods: 1. Real-time fluorescence quantitative PCR was used to detect the expression of miR-26a in neural stem cells and neuron cells. Use immunofluorescence staining and laser confocal method to detect and collect image of neuron cells. 3. The morphology of neurons was analyzed by Sholl with Image-ProPlus software, and the length and distribution of neurite processes were calculated. 4. Targetscan software was used to predict the target of miR-26a. 5. The specific overexpression of miR-26a was achieved by transfection of miR-26a mimic in neuron cells, and the specific inhibition of miR-26a was achieved by transfection of miR-26ainhibitor. The specific over-expression of phosphatase and tensin homolog (PTEN) was achieved by lentivirus infection, and the specific knock-down of PTEN was achieved by small interfering RNA (siRNA) using PTENshRNA. The expression level of PTEN protein was detected by Western blot. Results: the expression of 1.miR-26a in neuron cells was more abundant than that in neural stem cells. The number, length and distribution of neurite processes in over-expressed miR-26a group were significantly higher than those in control group. The number, length and distribution of neurite processes in the knockdown miR-26a group were significantly lower than those in the control group (n 100, P < 0.01). 3. The 3 'uncompiled region (3'UTR) of PTEN messenger RNA (mRNA) contained three miR-26a-specific binding sites. 4. The overexpression of miR-26a,PTEN protein decreased significantly in neuron cells. The number, length and distribution of neurite processes of over-expressed PTEN, neurons decreased significantly, while those of knockdown PTEN, neurons increased significantly (n100, P0.05). Conclusion: 1. Overexpression of miR-26a can promote the growth of neurite processes, increase the number, length and distribution of neurite processes, indicating that miR-26a plays an important role in neuronal morphological development. 2. PTEN is a key target of miR-26a in neuron cells. 3. Overexpression of PTEN gene can significantly inhibit the growth and development of neuron cells and decrease the plasticity of neuron cells. In neuron cells, PTEN is the key molecule that mediates miR-26a to play a related regulatory role. These findings provide a further understanding of neuronal plasticity and its growth and provide a new solution for Alzheimer's disease and its related neurodegenerative diseases.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

【共引文献】