高原低氧环境对骨髓造血干细胞增殖与定向选择分化的影响及其机制研究
发布时间：2018-08-12 20:03
【摘要】：目的:长期高原低氧环境生活会代偿性的引起机体红细胞生成增多。过度的红细胞生成会导致血液粘度增高,微循环阻力增加,加重组织细胞缺氧,并可引起一系列临床症状。既往研究虽然已经发现了低氧刺激机体生成高水平的促红细胞生成素(EPO)是高原红细胞增多的重要发生机制,但通过对高原生活人群的观察发现EPO的水平与红细胞的增生程度常常并不相关,提示存在非EPO途径和机制。 在成年机体,包括红细胞在内的所有类型成熟血细胞均起源于造血干细胞。研究发现造血干细胞参与了失血、感染等应激情况下的红细胞、白细胞生成过程,因此我们推测造血干细胞可能对高原低氧环境发生反应并在高原红细胞增生的过程中发挥调节作用。本课题针对这一问题展开研究。 方法:以模拟海拔6000m低氧环境暴露小鼠为模型,观察高原低氧环境暴露0,1,3,7,14,28天对骨髓与脾脏Lineage?Sca-1~+c-Kit~+ (LSK)表型造血干细胞的数量、增殖状况与定向分化系选择性的影响。在此基础上,将正常小鼠的骨髓LSK细胞置于不同的EPO浓度、不同的氧浓度下进行培养,或在培养体系中分别加入常氧和低氧暴露小鼠的骨髓培养上清,观察培养后Sca-1~+造血干细胞的增殖情况与定向分化潜能,以探讨机体高原低氧暴露时调控造血干细胞反应的可能机制。 结果:暴露于模拟海拔6000m低氧环境的小鼠骨髓和脾脏中lineage-Sca-1~+c-Kit~+ (LSK)表型造血干细胞数量均显著扩增,增加的造血干细胞既包括通常处于活跃细胞周期的短期重建造血能力造血干细胞(CD34+LSK细胞),也包括通常维持静息状态的长期重建造血能力造血干细胞(CD34-LSK细胞),低氧暴露时,骨髓和脾脏CD34+LSK细胞与CD34-LSK细胞的增殖率(BrdU掺入率)均显著增高,但细胞凋亡率无改变。 在小鼠低氧暴露的28天时间范围内,骨髓LSK造血干细胞在第3-28天呈现持续性的增殖,而脾脏造血干细胞只在第3-14天呈现暂时性的增殖,到第28天回落到低氧暴露前水平,即骨髓造血干细胞的变化时相性更符合高原红细胞长期慢性增多过程特点。另外,小鼠全身骨髓造血干细胞总数量约为脾脏造血干细胞的10倍,因此骨髓造血干细胞的增殖对高原红细胞增多可能发挥更大的作用。 通过对骨髓早期定向祖细胞的分析发现,在巨核-红系祖细胞(MEP)数量随着机体低氧暴露时间延长不断增多的同时,粒-单核祖细胞(GMP)数量在低氧暴露7-14天时出现暂时性的下降,通过观察排除了增殖和凋亡情况改变导致GMP减少的可能性,提示上游造血干细胞定向分化系选择性的变化导致了GMP的减少。对骨髓LSK造血干细胞红系和粒-单系定向分化潜能的直接观察结果证实了上述推测。一是低氧暴露小鼠骨髓LSK细胞红系特异性转录因子GATA-1的表达量增高,对应粒-单系特异性转录因子PU.1的表达量降低;二是低氧暴露小鼠的骨髓LSK细胞在体外半固体培养基上能形成较多比例的BFU-E红系集落和较少比例的CFU-GM粒巨噬系集落;三是低氧暴露小鼠的骨髓LSK细胞经红系诱导分化培养后能形成更多的Ter119+红系前体细胞。 通过将正常小鼠的骨髓LSK细胞在不同的EPO浓度、不同的O2浓度、或分别在培养体系中加入常氧与低氧暴露小鼠的骨髓培养上清进行培养得到以下结果: 第一、EPO水平的升高不影响培养后Sca-1~+造血干细胞的增殖与红系定向分化潜能。 第二、现有研究认为正常生理条件时骨髓多数造血干细胞的居留环境氧分压约相当于5%O2浓度,此氧分压可能随外界低氧环境进一步降低,但未观察到比5%O2更低的氧分压(2% O2)能促进培养后Sca-1~+造血干细胞的增殖和红系定向分化潜能。 第三、将低氧暴露小鼠的骨髓培养上清加入LSK细胞培养体系可以显著促进培养后Sca-1~+造血干细胞的增殖和红系定向分化潜能,通过抗体中和实验证明了低氧暴露后骨髓微环境中分泌增多的白介素3和白介素6参与了对造血干细胞的扩增作用,白介素3还参与了促进造血干细胞红系定向分化潜能。 结论:高原低氧环境下,一方面,骨髓造血干细胞通过增殖从数量上进行扩增和储备,以适应补充下游红细胞的需要;另一方面,骨髓造血干细胞更大比例的向红系选择性定向分化,使血细胞生成的优先性倾向于红系造血。上述两方面的调节促进了红系祖细胞(即EPO反应细胞)生成增多。骨髓微环境中多种造血生长因子的分泌变化可能参与了对造血干细胞数量和分化命运的调节。本研究发现的造血干细胞参与机制是一种非EPO依赖的高原红细胞增多新机制,将可能为高原红细胞增多症的防治提供新的思路和方向。
[Abstract]:OBJECTIVE: Long-term living in high altitude hypoxic environment will compensate for the increase of erythropoiesis. Excessive erythropoiesis will lead to increased blood viscosity, increased microcirculation resistance, aggravated tissue and cell hypoxia, and may cause a series of clinical symptoms. Previous studies have found that hypoxia stimulates the body to produce high levels of erythropoietin. Cytopoietin (EPO) is an important mechanism of erythrocyte hyperplasia at high altitude, but the observation of people living at high altitude shows that the level of EPO is not always related to the degree of erythrocyte hyperplasia, suggesting that there is a non-EPO pathway and mechanism.
In adult organisms, all types of mature blood cells, including red blood cells, originate from hematopoietic stem cells. Studies have shown that hematopoietic stem cells are involved in the process of erythrocyte and leukocyte formation under stress such as hemorrhage and infection. Therefore, we speculate that hematopoietic stem cells may react to altitude hypoxia and proliferate at altitude. In this process, we will play a regulatory role.
METHODS: Mice exposed to 6 000 m altitude hypoxia were used to observe the effects of 0,1,3,7,14,28 days exposure to high altitude hypoxia on the number, proliferation and selectivity of Lineage? Sca-1~+c-Kit~+ phenotypic hematopoietic stem cells (LSK) in bone marrow and spleen. The proliferation and directional differentiation potential of Sca-1~+ hematopoietic stem cells were observed in order to explore the possible mechanism of regulating the response of hematopoietic stem cells to hypoxic exposure at high altitude.
RESULTS: The number of lineage-Sca-1~+c-Kit~+ phenotypic hematopoietic stem cells (LSK) in bone marrow and spleen of mice exposed to simulated hypoxia at 6 000 m elevation was significantly increased, including both short-term reconstituted hematopoietic stem cells (CD34+LSK cells) normally in active cell cycles and normally resting state. The proliferative rate (BrdU incorporation rate) of CD34+LSK cells and CD34-LSK cells in bone marrow and spleen was significantly increased in long-term reconstituted hematopoietic stem cells (CD34-LSK cells) exposed to hypoxia, but the apoptosis rate remained unchanged.
In the 28-day period of hypoxic exposure in mice, bone marrow LSK hematopoietic stem cells proliferated continuously from day 3 to 28, while splenic hematopoietic stem cells proliferated temporarily from day 3 to 14, and then returned to the pre-hypoxic level on day 28, that is, the change of bone marrow hematopoietic stem cells was more consistent with the long-term chronic increase of red blood cells at high altitude. In addition, the total number of bone marrow hematopoietic stem cells in mice is about 10 times that of splenic hematopoietic stem cells, so the proliferation of bone marrow hematopoietic stem cells may play a greater role in Plateau erythrocytosis.
By analyzing the early directed progenitor cells in bone marrow, we found that the number of megakaryocyte-erythroid progenitor cells (MEP) increased with the prolongation of hypoxic exposure, and the number of granulocyte-monocyte progenitor cells (GMP) decreased temporarily after 7-14 days of hypoxic exposure. We ruled out that the change of proliferation and apoptosis might lead to the decrease of GMP. The direct observation of erythroid and granulocyte-monocyte differentiation potentials of bone marrow LSK hematopoietic stem cells confirms the above hypothesis. First, the expression of GATA-1, a erythroid-specific transcription factor, in bone marrow LSK cells of mice exposed to hypoxia increased, corresponding to granulocyte-monocyte. The expression of line-specific transcription factor PU.1 was decreased; the second was that the bone marrow LSK cells of hypoxic exposed mice could form more BFU-E erythroid colony and less CFU-GM granulocyte-macrophage colony on semi-solid medium in vitro; the third was that the bone marrow LSK cells of hypoxic exposed mice could form more Ter119 after erythroid-induced differentiation and culture. + erythroid precursor cells.
Normal mice bone marrow LSK cells were cultured at different EPO concentrations, O2 concentrations, or the supernatant of normal and hypoxic exposed mice bone marrow culture respectively.
Firstly, elevated EPO levels did not affect the proliferation and erythroid differentiation potential of cultured Sca-1~+ hematopoietic stem cells.
Secondly, existing studies suggest that the oxygen partial pressure of most hematopoietic stem cells in normal physiological conditions is about 5% O2, which may be further reduced with hypoxia, but no lower oxygen partial pressure (2% O2) than 5% O2 can promote the proliferation and erythroid differentiation potential of cultured Sca-1~+ hematopoietic stem cells.
Thirdly, adding the supernatant of bone marrow culture of hypoxic exposed mice to LSK cell culture system can significantly promote the proliferation and erythroid differentiation potential of cultured Sca-1~+ hematopoietic stem cells. The results of antibody neutralization demonstrated that the increased secretion of interleukin-3 and interleukin-6 in bone marrow microenvironment after hypoxic exposure participated in the expansion of hematopoietic stem cells. Interleukin 3 is also involved in promoting the differentiation potential of hematopoietic stem cells.
CONCLUSION: Under high altitude hypoxia, on the one hand, bone marrow hematopoietic stem cells proliferate and reserve in quantity to meet the needs of supplementing the downstream red blood cells; on the other hand, a larger proportion of bone marrow hematopoietic stem cells differentiate selectively into erythroid cells, which makes the priority of hematopoietic production tend to erythropoiesis. Hematopoietic progenitor cells (EPO-reactive cells) may be involved in the regulation of the number and differentiation fate of hematopoietic stem cells in the bone marrow microenvironment. The mechanism of hematopoietic stem cells involved in this study is a new mechanism of non-EPO-dependent plateau erythropoiesis, which may be the plateau. Prevention and treatment of polycythemia provide new ideas and directions.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363
本文编号：2180209
[Abstract]:OBJECTIVE: Long-term living in high altitude hypoxic environment will compensate for the increase of erythropoiesis. Excessive erythropoiesis will lead to increased blood viscosity, increased microcirculation resistance, aggravated tissue and cell hypoxia, and may cause a series of clinical symptoms. Previous studies have found that hypoxia stimulates the body to produce high levels of erythropoietin. Cytopoietin (EPO) is an important mechanism of erythrocyte hyperplasia at high altitude, but the observation of people living at high altitude shows that the level of EPO is not always related to the degree of erythrocyte hyperplasia, suggesting that there is a non-EPO pathway and mechanism.
In adult organisms, all types of mature blood cells, including red blood cells, originate from hematopoietic stem cells. Studies have shown that hematopoietic stem cells are involved in the process of erythrocyte and leukocyte formation under stress such as hemorrhage and infection. Therefore, we speculate that hematopoietic stem cells may react to altitude hypoxia and proliferate at altitude. In this process, we will play a regulatory role.
METHODS: Mice exposed to 6 000 m altitude hypoxia were used to observe the effects of 0,1,3,7,14,28 days exposure to high altitude hypoxia on the number, proliferation and selectivity of Lineage? Sca-1~+c-Kit~+ phenotypic hematopoietic stem cells (LSK) in bone marrow and spleen. The proliferation and directional differentiation potential of Sca-1~+ hematopoietic stem cells were observed in order to explore the possible mechanism of regulating the response of hematopoietic stem cells to hypoxic exposure at high altitude.
RESULTS: The number of lineage-Sca-1~+c-Kit~+ phenotypic hematopoietic stem cells (LSK) in bone marrow and spleen of mice exposed to simulated hypoxia at 6 000 m elevation was significantly increased, including both short-term reconstituted hematopoietic stem cells (CD34+LSK cells) normally in active cell cycles and normally resting state. The proliferative rate (BrdU incorporation rate) of CD34+LSK cells and CD34-LSK cells in bone marrow and spleen was significantly increased in long-term reconstituted hematopoietic stem cells (CD34-LSK cells) exposed to hypoxia, but the apoptosis rate remained unchanged.
In the 28-day period of hypoxic exposure in mice, bone marrow LSK hematopoietic stem cells proliferated continuously from day 3 to 28, while splenic hematopoietic stem cells proliferated temporarily from day 3 to 14, and then returned to the pre-hypoxic level on day 28, that is, the change of bone marrow hematopoietic stem cells was more consistent with the long-term chronic increase of red blood cells at high altitude. In addition, the total number of bone marrow hematopoietic stem cells in mice is about 10 times that of splenic hematopoietic stem cells, so the proliferation of bone marrow hematopoietic stem cells may play a greater role in Plateau erythrocytosis.
By analyzing the early directed progenitor cells in bone marrow, we found that the number of megakaryocyte-erythroid progenitor cells (MEP) increased with the prolongation of hypoxic exposure, and the number of granulocyte-monocyte progenitor cells (GMP) decreased temporarily after 7-14 days of hypoxic exposure. We ruled out that the change of proliferation and apoptosis might lead to the decrease of GMP. The direct observation of erythroid and granulocyte-monocyte differentiation potentials of bone marrow LSK hematopoietic stem cells confirms the above hypothesis. First, the expression of GATA-1, a erythroid-specific transcription factor, in bone marrow LSK cells of mice exposed to hypoxia increased, corresponding to granulocyte-monocyte. The expression of line-specific transcription factor PU.1 was decreased; the second was that the bone marrow LSK cells of hypoxic exposed mice could form more BFU-E erythroid colony and less CFU-GM granulocyte-macrophage colony on semi-solid medium in vitro; the third was that the bone marrow LSK cells of hypoxic exposed mice could form more Ter119 after erythroid-induced differentiation and culture. + erythroid precursor cells.
Normal mice bone marrow LSK cells were cultured at different EPO concentrations, O2 concentrations, or the supernatant of normal and hypoxic exposed mice bone marrow culture respectively.
Firstly, elevated EPO levels did not affect the proliferation and erythroid differentiation potential of cultured Sca-1~+ hematopoietic stem cells.
Secondly, existing studies suggest that the oxygen partial pressure of most hematopoietic stem cells in normal physiological conditions is about 5% O2, which may be further reduced with hypoxia, but no lower oxygen partial pressure (2% O2) than 5% O2 can promote the proliferation and erythroid differentiation potential of cultured Sca-1~+ hematopoietic stem cells.
Thirdly, adding the supernatant of bone marrow culture of hypoxic exposed mice to LSK cell culture system can significantly promote the proliferation and erythroid differentiation potential of cultured Sca-1~+ hematopoietic stem cells. The results of antibody neutralization demonstrated that the increased secretion of interleukin-3 and interleukin-6 in bone marrow microenvironment after hypoxic exposure participated in the expansion of hematopoietic stem cells. Interleukin 3 is also involved in promoting the differentiation potential of hematopoietic stem cells.
CONCLUSION: Under high altitude hypoxia, on the one hand, bone marrow hematopoietic stem cells proliferate and reserve in quantity to meet the needs of supplementing the downstream red blood cells; on the other hand, a larger proportion of bone marrow hematopoietic stem cells differentiate selectively into erythroid cells, which makes the priority of hematopoietic production tend to erythropoiesis. Hematopoietic progenitor cells (EPO-reactive cells) may be involved in the regulation of the number and differentiation fate of hematopoietic stem cells in the bone marrow microenvironment. The mechanism of hematopoietic stem cells involved in this study is a new mechanism of non-EPO-dependent plateau erythropoiesis, which may be the plateau. Prevention and treatment of polycythemia provide new ideas and directions.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363
【共引文献】
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1 衣龙燕;胡扬;聂晶;王景玲;;HiHiLo实施过程中网织红细胞与Hb变化规律的研究[J];中国体育科技;2010年03期
，本文编号：2180209
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