本文選題：低氧 + 神經(jīng)干細(xì)胞　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2010年碩士論文

【摘要】： 神經(jīng)干細(xì)胞(Neural Stem Cells,NSCs)由于其自我更新能力和多向分化潛能(能夠在體外分化成神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞),為神經(jīng)損傷和神經(jīng)系統(tǒng)退行性疾病的治療帶來了希望。既往的研究發(fā)現(xiàn)低氧環(huán)境能夠促進體外培養(yǎng)神經(jīng)干細(xì)胞的存活和增殖。但這種低氧的微環(huán)境是如何影響神經(jīng)干細(xì)胞增殖和代謝目前尚不知曉。因此,本實驗首先觀察了低氧對體外培養(yǎng)大鼠神經(jīng)干細(xì)胞增殖和代謝的影響。而在我們前期的研究中顯示,作為低氧環(huán)境中的最重要的調(diào)控分子之一的低氧誘導(dǎo)因子HIF-1(Hypoxia Inducible Factor-1)在低氧促神經(jīng)干細(xì)胞增殖中發(fā)揮著關(guān)鍵的調(diào)控作用。但是其具體機制仍有許多未明之處。 microRNA(miRNA)是一類21～23bp的內(nèi)源性單鏈RNA,參與調(diào)控了多種基因的表達,并在細(xì)胞增殖、分化和凋亡等多種生物學(xué)進程中起到非常重要的作用。目前對miRNAs在腫瘤細(xì)胞中的表達調(diào)控及功能作了大量的研究,作為腫瘤細(xì)胞所處的微環(huán)境,低氧與miRNAs的關(guān)聯(lián)也越來越引起人們的關(guān)注。那么,在低氧促神經(jīng)干細(xì)胞增殖過程中是否有miRNAs的參與?因此,在本研究的第二部分中我們利用芯片分析了體外培養(yǎng)的神經(jīng)干細(xì)胞中高表達的microRNAs,以及低氧對microRNAs表達變化的影響。并對在神經(jīng)細(xì)胞中高表達的miR-21在神經(jīng)干細(xì)胞中的作用進行了初步的探討。主要結(jié)果如下: 一.低氧對體外培養(yǎng)的大鼠胚胎中腦來源的神經(jīng)干細(xì)胞增殖和代謝的影響 1.大鼠胚胎中腦分離得到的神經(jīng)干細(xì)胞均為Nestin染色陽性,并可在體外誘導(dǎo)分化成神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞,說明我們成功地培養(yǎng)了神經(jīng)干細(xì)胞,且NSCs純度較高,可用于下面的實驗。 2.在低氧條件下,神經(jīng)干細(xì)胞培養(yǎng)第3、5d時,細(xì)胞的增殖明顯。表明低氧可促進神經(jīng)干細(xì)胞的增殖。神經(jīng)干細(xì)胞在低氧條件下培養(yǎng)后,代謝發(fā)生明顯的變化。隨著培養(yǎng)時間的延長,葡萄糖的利用增加,丙酮酸和乳酸的產(chǎn)生增加,培養(yǎng)液中pH降低,這種適度(輕微)降低的pH能促進暴露于低氧早期階段的NSCs的增殖。3.在低氧條件下,神經(jīng)干細(xì)胞的增殖增加,而細(xì)胞中ATP的產(chǎn)生在3 d、5 d時低于常氧組。低氧通過增加葡萄糖的轉(zhuǎn)運,以及糖酵解途徑中丙酮酸激酶的表達,來調(diào)節(jié)低氧環(huán)境中神經(jīng)干細(xì)胞的代謝發(fā)生變化。 二.miR-21的表達及對NSCs體外增殖的影響 在此部分我們利用非編碼RNA(ncRNAs)芯片篩選了一批在NSCs中特異表達和表達水平受低氧調(diào)控的非編碼RNA,并進一步對miR-21在低氧時的表達和在神經(jīng)干細(xì)胞中的作用進行了初步的探討。 1.用非編碼RNA(ncRNAs)芯片分析低氧促NSCs增殖過程中ncRNA表達的影響,這些RNA可能在神經(jīng)干細(xì)胞增殖中發(fā)揮了重要的作用。 2.首次發(fā)現(xiàn)miR-21的表達在神經(jīng)干細(xì)胞增殖過程中和細(xì)胞低氧、缺氧時的動態(tài)變化;在神經(jīng)干細(xì)胞中高表達的miR-21在3 %低氧條件下,miR-21的表達較常氧組有所降低,但在0.3 %缺氧條件下miR-21的表達卻明顯高于常氧組和3 %低氧組。提示miR-21在神經(jīng)干細(xì)胞的存活和增殖中可能具有一定的調(diào)節(jié)作用。 3.首次研究發(fā)現(xiàn)過表達miR-21可以促進體外培養(yǎng)的NSCs的存活和增殖,特別是可以提高神經(jīng)干細(xì)胞在缺氧時的存活能力。其調(diào)節(jié)的分子機制尚有待于進一步深入探討。 綜上所述,適度的低氧可促進體外培養(yǎng)的神經(jīng)干細(xì)胞的增殖;在低氧條件下,神經(jīng)干細(xì)胞的代謝發(fā)生明顯的變化,葡萄糖的利用明顯增加,主要通過糖酵解代謝途徑產(chǎn)生能量;低氧促進體外培養(yǎng)NSCs的增殖中,許多miRNAs的表達發(fā)生變化;其中在神經(jīng)干細(xì)胞中高表達的miR-21,可以促進神經(jīng)干細(xì)胞的存活和增殖,過表達后可以增加神經(jīng)干細(xì)胞在缺氧時的存活率。以上結(jié)果將為神經(jīng)干細(xì)胞的增殖調(diào)控和移植治療提供理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 馬洪巖;低氧對大鼠大腦皮層細(xì)胞凋亡及ROS、NO水平的影響[D];上海體育學(xué)院;2011年

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本文編號：2028743