發(fā)布時間：2018-08-12 20:03

【摘要】：目的:長期高原低氧環(huán)境生活會代償性的引起機(jī)體紅細(xì)胞生成增多。過度的紅細(xì)胞生成會導(dǎo)致血液粘度增高,微循環(huán)阻力增加,加重組織細(xì)胞缺氧,并可引起一系列臨床癥狀。既往研究雖然已經(jīng)發(fā)現(xiàn)了低氧刺激機(jī)體生成高水平的促紅細(xì)胞生成素(EPO)是高原紅細(xì)胞增多的重要發(fā)生機(jī)制,但通過對高原生活人群的觀察發(fā)現(xiàn)EPO的水平與紅細(xì)胞的增生程度常常并不相關(guān),提示存在非EPO途徑和機(jī)制。 在成年機(jī)體,包括紅細(xì)胞在內(nèi)的所有類型成熟血細(xì)胞均起源于造血干細(xì)胞。研究發(fā)現(xiàn)造血干細(xì)胞參與了失血、感染等應(yīng)激情況下的紅細(xì)胞、白細(xì)胞生成過程,因此我們推測造血干細(xì)胞可能對高原低氧環(huán)境發(fā)生反應(yīng)并在高原紅細(xì)胞增生的過程中發(fā)揮調(diào)節(jié)作用。本課題針對這一問題展開研究。 方法:以模擬海拔6000m低氧環(huán)境暴露小鼠為模型,觀察高原低氧環(huán)境暴露0,1,3,7,14,28天對骨髓與脾臟Lineage?Sca-1~+c-Kit~+ (LSK)表型造血干細(xì)胞的數(shù)量、增殖狀況與定向分化系選擇性的影響。在此基礎(chǔ)上,將正常小鼠的骨髓LSK細(xì)胞置于不同的EPO濃度、不同的氧濃度下進(jìn)行培養(yǎng),或在培養(yǎng)體系中分別加入常氧和低氧暴露小鼠的骨髓培養(yǎng)上清,觀察培養(yǎng)后Sca-1~+造血干細(xì)胞的增殖情況與定向分化潛能,以探討機(jī)體高原低氧暴露時調(diào)控造血干細(xì)胞反應(yīng)的可能機(jī)制。 結(jié)果:暴露于模擬海拔6000m低氧環(huán)境的小鼠骨髓和脾臟中l(wèi)ineage-Sca-1~+c-Kit~+ (LSK)表型造血干細(xì)胞數(shù)量均顯著擴(kuò)增,增加的造血干細(xì)胞既包括通常處于活躍細(xì)胞周期的短期重建造血能力造血干細(xì)胞(CD34+LSK細(xì)胞),也包括通常維持靜息狀態(tài)的長期重建造血能力造血干細(xì)胞(CD34-LSK細(xì)胞),低氧暴露時,骨髓和脾臟CD34+LSK細(xì)胞與CD34-LSK細(xì)胞的增殖率(BrdU摻入率)均顯著增高,但細(xì)胞凋亡率無改變。 在小鼠低氧暴露的28天時間范圍內(nèi),骨髓LSK造血干細(xì)胞在第3-28天呈現(xiàn)持續(xù)性的增殖,而脾臟造血干細(xì)胞只在第3-14天呈現(xiàn)暫時性的增殖,到第28天回落到低氧暴露前水平,即骨髓造血干細(xì)胞的變化時相性更符合高原紅細(xì)胞長期慢性增多過程特點。另外,小鼠全身骨髓造血干細(xì)胞總數(shù)量約為脾臟造血干細(xì)胞的10倍,因此骨髓造血干細(xì)胞的增殖對高原紅細(xì)胞增多可能發(fā)揮更大的作用。 通過對骨髓早期定向祖細(xì)胞的分析發(fā)現(xiàn),在巨核-紅系祖細(xì)胞(MEP)數(shù)量隨著機(jī)體低氧暴露時間延長不斷增多的同時,粒-單核祖細(xì)胞(GMP)數(shù)量在低氧暴露7-14天時出現(xiàn)暫時性的下降,通過觀察排除了增殖和凋亡情況改變導(dǎo)致GMP減少的可能性,提示上游造血干細(xì)胞定向分化系選擇性的變化導(dǎo)致了GMP的減少。對骨髓LSK造血干細(xì)胞紅系和粒-單系定向分化潛能的直接觀察結(jié)果證實了上述推測。一是低氧暴露小鼠骨髓LSK細(xì)胞紅系特異性轉(zhuǎn)錄因子GATA-1的表達(dá)量增高,對應(yīng)粒-單系特異性轉(zhuǎn)錄因子PU.1的表達(dá)量降低;二是低氧暴露小鼠的骨髓LSK細(xì)胞在體外半固體培養(yǎng)基上能形成較多比例的BFU-E紅系集落和較少比例的CFU-GM粒巨噬系集落;三是低氧暴露小鼠的骨髓LSK細(xì)胞經(jīng)紅系誘導(dǎo)分化培養(yǎng)后能形成更多的Ter119+紅系前體細(xì)胞。 通過將正常小鼠的骨髓LSK細(xì)胞在不同的EPO濃度、不同的O2濃度、或分別在培養(yǎng)體系中加入常氧與低氧暴露小鼠的骨髓培養(yǎng)上清進(jìn)行培養(yǎng)得到以下結(jié)果: 第一、EPO水平的升高不影響培養(yǎng)后Sca-1~+造血干細(xì)胞的增殖與紅系定向分化潛能。 第二、現(xiàn)有研究認(rèn)為正常生理條件時骨髓多數(shù)造血干細(xì)胞的居留環(huán)境氧分壓約相當(dāng)于5%O2濃度,此氧分壓可能隨外界低氧環(huán)境進(jìn)一步降低,但未觀察到比5%O2更低的氧分壓(2% O2)能促進(jìn)培養(yǎng)后Sca-1~+造血干細(xì)胞的增殖和紅系定向分化潛能。 第三、將低氧暴露小鼠的骨髓培養(yǎng)上清加入LSK細(xì)胞培養(yǎng)體系可以顯著促進(jìn)培養(yǎng)后Sca-1~+造血干細(xì)胞的增殖和紅系定向分化潛能,通過抗體中和實驗證明了低氧暴露后骨髓微環(huán)境中分泌增多的白介素3和白介素6參與了對造血干細(xì)胞的擴(kuò)增作用,白介素3還參與了促進(jìn)造血干細(xì)胞紅系定向分化潛能。 結(jié)論:高原低氧環(huán)境下,一方面,骨髓造血干細(xì)胞通過增殖從數(shù)量上進(jìn)行擴(kuò)增和儲備,以適應(yīng)補充下游紅細(xì)胞的需要;另一方面,骨髓造血干細(xì)胞更大比例的向紅系選擇性定向分化,使血細(xì)胞生成的優(yōu)先性傾向于紅系造血。上述兩方面的調(diào)節(jié)促進(jìn)了紅系祖細(xì)胞(即EPO反應(yīng)細(xì)胞)生成增多。骨髓微環(huán)境中多種造血生長因子的分泌變化可能參與了對造血干細(xì)胞數(shù)量和分化命運的調(diào)節(jié)。本研究發(fā)現(xiàn)的造血干細(xì)胞參與機(jī)制是一種非EPO依賴的高原紅細(xì)胞增多新機(jī)制,將可能為高原紅細(xì)胞增多癥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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R363

【共引文獻(xiàn)】

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1 衣龍燕;胡揚;聶晶;王景玲;;HiHiLo實施過程中網(wǎng)織紅細(xì)胞與Hb變化規(guī)律的研究[J];中國體育科技;2010年03期

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本文編號：2180209