低氧对神经干细胞增殖和代谢的影响及miR-21的表达和作用的研究

发布时间：2018-06-17 00:29

本文选题：低氧 + 神经干细胞　；参考：《中国人民解放军军事医学科学院》2010年硕士论文

【摘要】： 神经干细胞(Neural Stem Cells,NSCs)由于其自我更新能力和多向分化潜能(能够在体外分化成神经元、星形胶质细胞和少突胶质细胞),为神经损伤和神经系统退行性疾病的治疗带来了希望。既往的研究发现低氧环境能够促进体外培养神经干细胞的存活和增殖。但这种低氧的微环境是如何影响神经干细胞增殖和代谢目前尚不知晓。因此,本实验首先观察了低氧对体外培养大鼠神经干细胞增殖和代谢的影响。而在我们前期的研究中显示,作为低氧环境中的最重要的调控分子之一的低氧诱导因子HIF-1(Hypoxia Inducible Factor-1)在低氧促神经干细胞增殖中发挥着关键的调控作用。但是其具体机制仍有许多未明之处。 microRNA(miRNA)是一类21～23bp的内源性单链RNA,参与调控了多种基因的表达,并在细胞增殖、分化和凋亡等多种生物学进程中起到非常重要的作用。目前对miRNAs在肿瘤细胞中的表达调控及功能作了大量的研究,作为肿瘤细胞所处的微环境,低氧与miRNAs的关联也越来越引起人们的关注。那么,在低氧促神经干细胞增殖过程中是否有miRNAs的参与?因此,在本研究的第二部分中我们利用芯片分析了体外培养的神经干细胞中高表达的microRNAs,以及低氧对microRNAs表达变化的影响。并对在神经细胞中高表达的miR-21在神经干细胞中的作用进行了初步的探讨。主要结果如下: 一.低氧对体外培养的大鼠胚胎中脑来源的神经干细胞增殖和代谢的影响 1.大鼠胚胎中脑分离得到的神经干细胞均为Nestin染色阳性,并可在体外诱导分化成神经元、星形胶质细胞和少突胶质细胞,说明我们成功地培养了神经干细胞,且NSCs纯度较高,可用于下面的实验。 2.在低氧条件下,神经干细胞培养第3、5d时,细胞的增殖明显。表明低氧可促进神经干细胞的增殖。神经干细胞在低氧条件下培养后,代谢发生明显的变化。随着培养时间的延长,葡萄糖的利用增加,丙酮酸和乳酸的产生增加,培养液中pH降低,这种适度(轻微)降低的pH能促进暴露于低氧早期阶段的NSCs的增殖。3.在低氧条件下,神经干细胞的增殖增加,而细胞中ATP的产生在3 d、5 d时低于常氧组。低氧通过增加葡萄糖的转运,以及糖酵解途径中丙酮酸激酶的表达,来调节低氧环境中神经干细胞的代谢发生变化。二.miR-21的表达及对NSCs体外增殖的影响在此部分我们利用非编码RNA(ncRNAs)芯片筛选了一批在NSCs中特异表达和表达水平受低氧调控的非编码RNA,并进一步对miR-21在低氧时的表达和在神经干细胞中的作用进行了初步的探讨。 1.用非编码RNA(ncRNAs)芯片分析低氧促NSCs增殖过程中ncRNA表达的影响,这些RNA可能在神经干细胞增殖中发挥了重要的作用。 2.首次发现miR-21的表达在神经干细胞增殖过程中和细胞低氧、缺氧时的动态变化;在神经干细胞中高表达的miR-21在3 %低氧条件下,miR-21的表达较常氧组有所降低,但在0.3 %缺氧条件下miR-21的表达却明显高于常氧组和3 %低氧组。提示miR-21在神经干细胞的存活和增殖中可能具有一定的调节作用。 3.首次研究发现过表达miR-21可以促进体外培养的NSCs的存活和增殖,特别是可以提高神经干细胞在缺氧时的存活能力。其调节的分子机制尚有待于进一步深入探讨。综上所述,适度的低氧可促进体外培养的神经干细胞的增殖;在低氧条件下,神经干细胞的代谢发生明显的变化,葡萄糖的利用明显增加,主要通过糖酵解代谢途径产生能量;低氧促进体外培养NSCs的增殖中,许多miRNAs的表达发生变化;其中在神经干细胞中高表达的miR-21,可以促进神经干细胞的存活和增殖,过表达后可以增加神经干细胞在缺氧时的存活率。以上结果将为神经干细胞的增殖调控和移植治疗提供理论基础。
[Abstract]:Neural Stem Cells (NSCs), due to its self renewal capacity and pluripotent differentiation potential (capable of differentiation into neurons, astrocytes and oligodendrocytes in vitro), has brought hope for the treatment of nerve damage and neurodegenerative diseases. But it is not known how the hypoxia microenvironment affects the proliferation and metabolism of neural stem cells. Therefore, this experiment first observed the effect of hypoxia on the proliferation and metabolism of rat neural stem cells in vitro. In our previous study, it was shown as the most important regulator in the hypoxic environment. One of the hypoxia inducible factor HIF-1 (Hypoxia Inducible Factor-1) plays a key role in the proliferation of hypoxic neural stem cells. However, there are still many unknown mechanisms in its specific mechanism.
MicroRNA (miRNA) is a kind of endogenous single strand RNA from 21 to 23bp. It plays a very important role in many biological processes, such as cell proliferation, differentiation and apoptosis. At present, the regulation and function of miRNAs expression in tumor cells have been studied as a microenvironment of tumor cells. The association of hypoxia with miRNAs has attracted more and more attention. Then, is there a miRNAs involvement in the proliferation of hypoxia - induced neural stem cells? So, in the second part of this study, we used the chip to analyze the high expression of microRNAs in the cultured neural stem cells, and the effect of hypoxia on the changes of microRNAs expression. The role of miR-21, which is highly expressed in nerve cells, in neural stem cells was preliminarily discussed. The main results are as follows:
Effects of hypoxia on the proliferation and metabolism of neural stem cells derived from rat mesencephalon in vitro
The neural stem cells isolated from the middle brain of the 1. rat embryos were all positive for Nestin staining, and could be induced to differentiate into neurons, astrocytes and oligodendrocytes in vitro, indicating that we successfully cultured neural stem cells, and the purity of NSCs is high, which can be used in the following experiments.
2. under the condition of hypoxia, the proliferation of the cells was obvious when the neural stem cells were cultured at 3,5d. It showed that hypoxia could promote the proliferation of neural stem cells. After the culture of hypoxia, the metabolism of neural stem cells changed obviously. With the prolongation of the culture time, the use of glucose increased, the production of pyruvic acid and lactic acid increased, and the pH in the culture medium decreased. This moderate (slightly) reduced pH promotes the proliferation of NSCs, which is exposed to the early stage of hypoxia, and increases the proliferation of neural stem cells under hypoxic conditions, while the production of ATP in the cells is lower than that of the normal oxygen group at 3 D and 5 d. Hypoxia can regulate the hypoxia environment by increasing the transport of glucose and the expression of pyruvate kinase in glycolysis pathway. The metabolism of neural stem cells changes.
Expression of two.MiR-21 and its effect on NSCs proliferation in vitro
In this part, we used non coded RNA (ncRNAs) chips to screen a group of non coded RNA specific expression and expression level regulated by hypoxia in NSCs, and further discussed the expression of miR-21 in hypoxia and the role of miR-21 in neural stem cells.
1. the non coded RNA (ncRNAs) chips were used to analyze the effect of ncRNA expression during the proliferation of hypoxia promoting NSCs, and these RNA may play an important role in the proliferation of neural stem cells.
2. the expression of miR-21 was found for the first time in the proliferation of neural stem cells and in the dynamic changes of hypoxia and hypoxia. The expression of high expression of miR-21 in neural stem cells was lower than that of the normal oxygen group under 3% hypoxia conditions, but the expression of miR-21 was significantly higher than that of the normal oxygen group and the 3% hypoxic group under the condition of 0.3% hypoxia. The expression of miR-21 was significantly higher than that of the normal oxygen group and the 3% hypoxia group. It may play a regulatory role in the survival and proliferation of neural stem cells.
3. the first study found that the expression of miR-21 can promote the survival and proliferation of NSCs in vitro, especially the survival ability of neural stem cells in anoxia. The molecular mechanism of the regulation remains to be further explored.
To sum up, moderate hypoxia can promote the proliferation of neural stem cells in vitro; in hypoxic conditions, the metabolism of neural stem cells obviously changes, the use of glucose is obviously increased, and energy is produced mainly through glycolytic pathway; hypoxia promotes the proliferation of NSCs in vitro, and the expression of many miRNAs changes; The high expression of miR-21 in neural stem cells can promote the survival and proliferation of neural stem cells. After overexpression, the survival rate of neural stem cells can be increased in hypoxia. The above results will provide a theoretical basis for the regulation of proliferation and transplantation of neural stem cells.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【引证文献】