四种miRNA在神经干细胞与运动神经元以及神经干细胞分化过程中的表达研究

发布时间：2018-09-18 19:16

【摘要】： 研究背景：BMSCs是成体非神经组织源性神经干细胞研究最多的一种。目前对于BMSCs定向分化为神经元的分子调控机制仍不清楚,因为miR-124和miR-128在神经系统含量丰富且特异性较高,本实验通过体外诱导BMSCs向神经元分化,观察miR-124和miR-128的表达情况,以了解它们对BMSCs定向分化为神经元过程中发挥的作用。目的：本部分的研究旨在通过检测BMSCs诱导分化为神经元的过程中miR-124和miR-128的表达情况,从而初步探讨miR-124和miR-128在BMSCs诱导分化为神经元的过程中所发挥的作用。方法：采用全骨髓培养法体外分离培养获得BMSCs,根据培养细胞的贴壁能力进行纯化,倒置显微镜下观察其形态变化,取传代培养至第三代的BMSCs,其中一部分细胞进行CD71免疫荧光技术检测,另一部分细胞加入诱导分化培养液进行诱导分化。然后应用ABI公司的TaqMan MicroRNA Assays real-time PCR技术,采用两步法,先将总RNA逆转录为cDNA,再检测miR-124和miR-128在BMSCs诱导分化过程中的表达变化。结果：miR-124在BMSCs分化所得神经元中的表达是未分化时的0.051倍(P0.05)；miR-128在BMSCs分化所得神经元中的表达是未分化时的0.070倍(P0.05)。结论：miR-124和miR-128在BMSCs诱导分化为神经元的过程中可起重着要的调控作用。研究背景：神经干细胞是一群具有自我更新、自我增殖和多向分化潜能的细胞。NSCs可定向诱导分化为胆碱能神经元；Hox是运动神经元(motor neurons, MNs)在体内发育的关键因子之一,而miR-126可以通过绑定到同源框来调控Hoxa9,因此,我们通过实验来探讨是否miR-126在脊髓运动神经元的发育过程也起相关作用。miR-31是一种胚胎干细胞特异性的niRNA。我们通过培养、分离和诱导脊髓源神经干细胞(spinal cord stem cells, SCSCs)以及采用免疫磁珠分选法分离纯化胚胎脊髓源运动神经元,然后检测miR-126和miR-31在MNs及SCSCs分化过程中的表达情况,从而初步探讨它们在神经干细胞中定向分化过程中的作用。目的：通过观察miR-126和miR-31在MNs及神经干细胞分化过程中的表达情况,初步探讨它们在神经干细胞定向分化过程中的作用。方法：采用孕龄17d的胚胎大鼠脊髓组织,分离、培养神经干细胞,传代扩增。对第3代的神经干细胞球进行神经上皮干细胞蛋白(neuroepithelial stem proteins, Nestin)免疫荧光检测。然后,应用诱导因子RA、音猬因子(sonic hedgehog, Shh)、NT-3和BDNF对神经干细胞进行诱导分化。分化两周后,用免疫荧光检测胆碱能神经元特异性标志物胆碱乙酰转移酶(choline acetyltransferase, ChAT)的表达。在另外一个实验中,我们用免疫磁珠法分离纯化胚胎脊髓源运动神经元。最后应用ABI公司的TaqMan MicroRNA Assays real-time PCR技术,来观察miR-126和miR-31在MNs及神经干细胞分化过程中的表达情况。结果：miR-126在SCSCs中的表达是其分化所得胆碱能神经元中的0.142倍。miR-31在SCSCs中的表达是其分化所得胆碱能神经元的0.118倍。miR-126在胆碱能神经元中的表达是在MNs中的0.002倍。miR-31在胆碱能神经元中的表达是在MNs中的56.444倍。Hoxa9在胆碱能神经元中的表达是在MNs中的0.169倍。Shh在MNs中有表达,而在胆碱能神经元中用实时定量PCR无法检测到。结论：miR-31在胆碱能神经元中较运动神经元高表达说明了我们诱导所得细胞(胆碱能神经元)与目的细胞(运动神经元)之间可能存在差异；miR-126和miR-31在神经干细胞的分化过程中起着重要的调控重要作用；
[Abstract]:BACKGROUND: BMSCs are one of the most widely studied adult non-neural tissue-derived neural stem cells. At present, the molecular mechanism of directional differentiation of BMSCs into neurons is still unclear, because of the rich and high specificity of microRNAs-124 and microRNAs-128 in the nervous system. This experiment induced BMSCs to differentiate into neurons in vitro, and observed microRNAs-124 and microRNAs-128. The expression of iR-128 in order to understand their role in BMSCs directed differentiation into neurons.
AIM: To investigate the role of microRNAs-124 and microRNAs-128 in BMSCs-induced neuronal differentiation by detecting the expression of microRNAs-124 and microRNAs-128 during BMSCs-induced neuronal differentiation.
METHODS: BMSCs were isolated and cultured in vitro by whole bone marrow culture method. BMSCs were purified according to the adherence ability of the cultured cells. The morphological changes were observed under inverted microscope. BMSCs were subcultured to the third generation. Some of the cells were detected by CD71 immunofluorescence technique, and the others were induced by adding induced differentiation medium. Differentiation. Then, using the TaqMan MicroRNA Assay real-time PCR technique of ABI Company, the total RNA was first retrieved into a cDNA by two-step method, and then the expression changes of microRNA-124 and microRNA-128 in BMSCs induced differentiation were detected.
Results: The expression of Mi-124 in differentiated BMSCs neurons was 0.051 times higher than that in undifferentiated BMSCs neurons (P 0.05), and the expression of Mi-128 in differentiated BMSCs neurons was 0.070 times higher than that in undifferentiated BMSCs neurons (P 0.05).
Conclusion: Mi-124 and Mi-128 play important roles in the differentiation of BMSCs into neurons.
BACKGROUND: Neural stem cells are a group of cells with the potential of self-renewal, self-proliferation and multi-differentiation. NSCs can induce the differentiation of cholinergic neurons directionally; Hox is one of the key factors for the development of motor neurons (MNs) in vivo, and Mi-126 can regulate Hoxa9 by binding to homologous frameworks. Therefore, we pass on Mi-31 is an embryonic stem cell-specific niRNA. We isolate and induce spinal cord stem cells (SCSCs) by culture and isolate and purify embryonic spinal cord-derived motor nerves by immunomagnetic beads sorting. We then examined the expression of microRNAs-126 and microRNAs-31 in the differentiation of NN and SCSCs to explore their roles in the differentiation of neural stem cells.
Objective: To investigate the expression of microRNAs-126 and microRNAs-31 in the differentiation of neural stem cells (NSCs) and neural stem cells (NSCs).
METHODS: Neural stem cells (NSCs) were isolated from spinal cord of 17-day-old embryonic rats and cultured for passage and amplification. Neuroepithelial stem cell proteins (Nestin) of the third generation NSCs were detected by immunofluorescence assay. Then, RA, Shh, NT-3 and BDNF were applied to the neurons. Stem cells were induced to differentiate. Two weeks after differentiation, the expression of choline acetyltransferase (ChAT), a specific marker of cholinergic neurons, was detected by immunofluorescence assay. In another experiment, we isolated and purified embryonic spinal cord-derived motor neurons by immunomagnetic beads. Finally, ABI's TaqMan MicroRNA As was used. To observe the expression of microRNAs-126 and microRNAs-31 in NN and neural stem cell differentiation by real-time PCR.
Results: The expression of Mi-126 in SCSC was 0.142 times as high as that in differentiated cholinergic neurons. The expression of Mi-31 in SCSC was 0.118 times as high as that in differentiated cholinergic neurons. The expression of Mi-126 in cholinergic neurons was 0.002 times as high as that in MNS. The expression of Mi-31 in cholinergic neurons was 56.444 times as high as that in MNS. The expression of cholinergic neurons was 0.169 times that of MNs. Shh was expressed in MNs, but could not be detected in cholinergic neurons by real-time quantitative PCR.
CONCLUSION: The high expression of microRNAs-31 in cholinergic neurons suggests that there may be differences between the cholinergic neurons and the target cells, and microRNAs-126 and microRNAs-31 play an important role in the differentiation of neural stem cells.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【相似文献】