低氧促进人骨髓间充质干细胞增殖和分化及其机制研究

发布时间：2018-01-10 15:04

本文关键词：低氧促进人骨髓间充质干细胞增殖和分化及其机制研究　出处：《中国人民解放军军事医学科学院》2005年博士论文　论文类型：学位论文

【摘要】：氧是维持生命的必要条件,也是细胞功能的一种重要生理调节因子,而低氧是生命发育的基本环境。例如,在妊娠的早期,由于没有血管的形成,所以胚胎是在低氧环境中发育的。低氧作为一种生理性的刺激因素,影响着胚胎的发生、发育及正常功能的维持。低氧可激活低氧诱导因子(HIF),从而调控一系列与低氧相关基因的表达。例如,诱导与葡萄糖分解、转运、红细胞和血管再生等相关的基因,使其表达增加,从而维持体内氧环境的稳态。关于低氧作用的研究,目前多集中在低氧对细胞的损伤和有关的适应机制方面,而对低氧在细胞增殖、分化中影响的研究较少。特别是对于体内胚胎发生时期和成年后的一些生理、病理过程中,出现细胞局部微环境低氧的情况重视不足。干细胞包括骨髓间充质干细胞(MSCs)体外培养和增殖分化作用的研究都是在常氧(20%O_2)条件下进行的,这样难以反映体内的真实情况。低氧在体内作为一种生理性的刺激因素,对体外培养的MSCs增殖和分化的作用目前还不清楚,也没有引起科研人员的足够重视。而人骨髓间充质干细胞(hMSCs)是目前临床应用前景最好的成体干细胞,因此,本文就低氧对hMSCs体外增殖和分化作用进行了初步研究。 1.低氧对hMSCs体外增殖的作用探讨不同方式低氧对hMSCs体外增殖的影响。实验采用血球计数板计数法和流式细胞术分别观察了间歇性低氧(3%O_2、10%O_2)和持续性低氧(3%O_2、10%O_2、100μmol/L CoCl_2、200μmol/L CoCl_2)对hMSCs数量以及增殖指数的影响。结果发现,间歇性低氧对hMSCs数量和增殖指数无明显影响;持续性低氧各组hMSCs数量和增殖指数均增加(P0.05)。提示持续性低氧可促进体外培养的hMSCs增殖,说明持续性低氧作为体外的一种可控制因素,可调节hMSCs的体外增殖,对于hMSCs的临床应用具有一定的意义。同时本研究还应用基因芯片技术分析了低氧对hMSCs增殖过程中基因表达谱的变化。结果显示21329个基因中282个基因有差异性表达。从基因学的角度证实了低氧对hMSCs增殖的影响是一个多基因参与的复杂过程。并从基因芯片的结果中筛选出5个目的基因,用RT-PCR方法检测了这5个基因的表达变化。发现在
[Abstract]:Oxygen is a necessary condition for life and an important physiological regulator of cell function, and hypoxia is the basic environment for life development. For example, in the early stage of pregnancy, there is no blood vessel formation. Therefore, embryos are developed in hypoxic environment. As a physiological stimulant, hypoxia affects the development, development and maintenance of normal function of embryos. Hypoxia can activate hypoxia inducible factor (HIFs). It regulates the expression of a series of genes related to hypoxia, such as inducing and increasing the expression of genes related to glucose decomposition, transport, erythrocyte and vascular regeneration. So as to maintain the homeostasis of oxygen environment in vivo. The current research on hypoxic effect mainly focuses on the damage of hypoxia to cells and related adaptive mechanisms, and on the proliferation of hypoxia in cells. There are few studies on the effects of differentiation, especially in some physiological and pathological processes during embryogenesis and adulthood in vivo. Insufficient attention was paid to the occurrence of hypoxia in the local microenvironment. Stem cells, including bone marrow mesenchymal stem cells (MSCs), were cultured and differentiated in vitro. Under conditions. As a physiological stimulant in vivo, the effect of hypoxia on the proliferation and differentiation of MSCs cultured in vitro is still unclear. Human bone marrow mesenchymal stem cells (hMSCs) are currently the most promising adult stem cells in clinical application. The effects of hypoxia on proliferation and differentiation of hMSCs in vitro were studied. 1. Effects of hypoxia on proliferation of hMSCs in vitro To investigate the effect of hypoxia on the proliferation of hMSCs in vitro, the intermittent hypoxia of 3O 2 was observed by blood cell counter counting and flow cytometry, respectively. 10 / 10 mol/L / CoCl_2 / s / / /\\\. The effects of 200 渭 mol/L CoCl2) on the number of hMSCs and proliferation index were observed. The results showed that intermittent hypoxia had no significant effect on the number and proliferation index of hMSCs. The number and proliferation index of hMSCs were increased in the groups of persistent hypoxia, indicating that persistent hypoxia could promote the proliferation of hMSCs in vitro. It is suggested that persistent hypoxia, as a controllable factor in vitro, can regulate the proliferation of hMSCs in vitro, and has a certain significance for the clinical application of hMSCs. At the same time, gene microarray technique was used to analyze the changes of gene expression profile during hypoxia on hMSCs proliferation. The results showed that 282 genes were differentially expressed in 21329 genes. The effect of hypoxia on the proliferation of hMSCs was proved to be a complex process in which multiple genes were involved and five target genes were screened from the results of gene microarray. The expression changes of these five genes were detected by RT-PCR.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329

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