G-CSF诱导小鼠造血干细胞动员过程中成骨细胞及破骨细胞的变化
发布时间：2018-09-05 17:15
【摘要】：目的:造血干细胞(Hematopoietic stem cell HSC)目前被广泛用于移植治疗恶性血液系统疾病,采集足够量的造血干细胞是移植治疗成功的关键前提,近年研究明确,骨髓微环境中存在着两种以上的骨髓龛,其中骨内膜龛对维持造血干细胞的稳定静止起着重要作用,而成骨细胞是骨内膜龛的重要组成部分,大量实验表明,G-CSF动员后成骨细胞数量减少,功能减低,破骨细胞作为与成骨细胞相反作用的细胞,在动员过程中发生什么变化,值得我们去研究,本研究以小鼠为研究对象,观察G-CSF动员对小鼠成骨、破骨细胞的影响,以及通过PTH诱导激活破骨细胞,观察对造血干细胞动员的影响。方法:利用流式细胞计数检测动员0、3、5天小鼠外周血干细胞比例,观察G-CSF诱导动员的情况,通过实时定量-多聚酶链式反应(RQ-PCR)检测动员前后骨髓细胞骨钙素(Osteocalcin OCN)、基质细胞衍生因子-1(Stromal cell-derived factor-1 SDF-1)和基质金属蛋白酶9(Matrix metalloproteinases9 MMP9)的m RNA表达,免疫组织化学检测骨髓OCN(成骨细胞特异性表达)阳性细胞的数量来比较动员前后成骨细胞的数量和功能上的变化,ELISA法检测小鼠周血TRACP-5b的浓度反应破骨细胞活性,抗酒石酸磷酸酶(tartrate-resistant acid phosphatase TRAP)染色检测小鼠股骨中破骨细胞数量,评价G-CSF动员对成骨、破骨细胞的影响。大剂量PTH(Parathyroid hormone甲状旁腺素)激活破骨细胞,观察激活破骨细胞后能否产生HSC动员效应。结果:1.骨髓SDF-1和OCN表达减少,提示成骨细胞功能降低;MMP9表达增高,提示中性粒细胞释放MMP9,骨髓基质发生蛋白水解。2.显微镜下观察动员后骨髓成骨细胞数量减少,形态由原来卵圆形变为梭形至扁平消失。3.小鼠外周血TRACP-5b增多,0天(4.43±0.52IU/L)、5天(10.49±1.75m IU/m L),P0.05,提示破骨细胞活性增高;TRAP染色提示注射G-CSF后破骨细胞数量增多。4.大剂量PTH激活破骨细胞能够产生HSC动员效应。结论G-CSF动员后成骨细胞数量及活性降低,导致SDF-1趋化能力减弱,破坏造血干细胞静止状态引发动员;破骨细胞激活后可能与成骨细胞相互作用影响动员发生。
[Abstract]:Objective: hematopoietic stem cell (Hematopoietic stem cell HSC) has been widely used in the treatment of malignant hematological diseases. The acquisition of sufficient amount of hematopoietic stem cells is the key prerequisite for the successful treatment of hematopoietic stem cells. There are more than two kinds of bone marrow niches in bone marrow microenvironment, in which endomembrane niches play an important role in maintaining the stability of hematopoietic stem cells, and osteoblasts are important components of osteoblast niches. A large number of experiments showed that the number and function of osteoblasts decreased after G-CSF mobilization. The changes of osteoclasts in the mobilization process of osteoclasts, which are opposite to those of osteoblasts, are worthy of our study. To observe the effect of G-CSF mobilization on osteogenesis and osteoclast in mice, and to observe the effect of PTH on the mobilization of hematopoietic stem cells. Methods: the percentage of peripheral blood stem cells (PBSCs) in mice mobilized for 3 days was detected by flow cytometry, and the mobilization induced by G-CSF was observed. The expression of osteocalcin (Osteocalcin OCN), stromal cell derived factor 1 (Stromal cell-derived factor-1 SDF-1) and matrix metalloproteinase 9 (Matrix metalloproteinases9 MMP9) in bone marrow cells before and after mobilization was detected by real-time quantification-polymerase chain reaction (RQ-PCR). The number of bone marrow OCN (osteoblast specific expression) positive cells was detected by immunohistochemistry to compare the changes in the number and function of osteoblasts before and after mobilization. Elisa was used to detect the concentration of TRACP-5b in peripheral blood of mice, which reflected the osteoclast activity. Anti-tartrate phosphatase (tartrate-resistant acid phosphatase TRAP) staining was used to detect the number of osteoclasts in the femur of mice and to evaluate the effect of G-CSF mobilization on osteoblasts and osteoclasts. High dose PTH (Parathyroid hormone parathyroid hormone (PTH (Parathyroid hormone) activated osteoclasts to observe whether HSC mobilization could be produced after activation of osteoclasts. The result is 1: 1. The decreased expression of SDF-1 and OCN in bone marrow suggested that the function of osteoblasts decreased and the expression of MMP9 increased, suggesting that neutrophils released MMP9, bone marrow stromal proteolysis. 2. Under microscope, the number of bone marrow osteoblasts decreased and the shape of bone marrow osteoblasts changed from oval to fusiform to flat disappearance. 3. The increase of TRACP-5b in peripheral blood of mice was (4.43 卤0.52IU/L) / 5d (10.49 卤1.75m IU/m / L) P0.05, suggesting that the increased activity of osteoclasts and trap staining suggested that the number of osteoclasts increased after G-CSF injection. High dose PTH activates osteoclasts and produces HSC mobilization. Conclusion the number and activity of osteoblasts decreased after G-CSF mobilization, which resulted in the decrease of chemotactic ability of SDF-1, which resulted in the mobilization of osteoclasts after the activation of osteoclasts, and the activation of osteoclasts may influence the mobilization of osteoblasts.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7
本文编号：2224913
[Abstract]:Objective: hematopoietic stem cell (Hematopoietic stem cell HSC) has been widely used in the treatment of malignant hematological diseases. The acquisition of sufficient amount of hematopoietic stem cells is the key prerequisite for the successful treatment of hematopoietic stem cells. There are more than two kinds of bone marrow niches in bone marrow microenvironment, in which endomembrane niches play an important role in maintaining the stability of hematopoietic stem cells, and osteoblasts are important components of osteoblast niches. A large number of experiments showed that the number and function of osteoblasts decreased after G-CSF mobilization. The changes of osteoclasts in the mobilization process of osteoclasts, which are opposite to those of osteoblasts, are worthy of our study. To observe the effect of G-CSF mobilization on osteogenesis and osteoclast in mice, and to observe the effect of PTH on the mobilization of hematopoietic stem cells. Methods: the percentage of peripheral blood stem cells (PBSCs) in mice mobilized for 3 days was detected by flow cytometry, and the mobilization induced by G-CSF was observed. The expression of osteocalcin (Osteocalcin OCN), stromal cell derived factor 1 (Stromal cell-derived factor-1 SDF-1) and matrix metalloproteinase 9 (Matrix metalloproteinases9 MMP9) in bone marrow cells before and after mobilization was detected by real-time quantification-polymerase chain reaction (RQ-PCR). The number of bone marrow OCN (osteoblast specific expression) positive cells was detected by immunohistochemistry to compare the changes in the number and function of osteoblasts before and after mobilization. Elisa was used to detect the concentration of TRACP-5b in peripheral blood of mice, which reflected the osteoclast activity. Anti-tartrate phosphatase (tartrate-resistant acid phosphatase TRAP) staining was used to detect the number of osteoclasts in the femur of mice and to evaluate the effect of G-CSF mobilization on osteoblasts and osteoclasts. High dose PTH (Parathyroid hormone parathyroid hormone (PTH (Parathyroid hormone) activated osteoclasts to observe whether HSC mobilization could be produced after activation of osteoclasts. The result is 1: 1. The decreased expression of SDF-1 and OCN in bone marrow suggested that the function of osteoblasts decreased and the expression of MMP9 increased, suggesting that neutrophils released MMP9, bone marrow stromal proteolysis. 2. Under microscope, the number of bone marrow osteoblasts decreased and the shape of bone marrow osteoblasts changed from oval to fusiform to flat disappearance. 3. The increase of TRACP-5b in peripheral blood of mice was (4.43 卤0.52IU/L) / 5d (10.49 卤1.75m IU/m / L) P0.05, suggesting that the increased activity of osteoclasts and trap staining suggested that the number of osteoclasts increased after G-CSF injection. High dose PTH activates osteoclasts and produces HSC mobilization. Conclusion the number and activity of osteoblasts decreased after G-CSF mobilization, which resulted in the decrease of chemotactic ability of SDF-1, which resulted in the mobilization of osteoclasts after the activation of osteoclasts, and the activation of osteoclasts may influence the mobilization of osteoblasts.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7
【参考文献】
相关期刊论文 前3条
1 Mengrui Wu;Guiqian Chen;Yi-Ping Li;;TGF-β and BMP signaling in osteoblast,skeletal development,and bone formation,homeostasis and disease[J];Bone Research;2016年01期
2 Takeshi Miyamoto;;Role of osteoclasts in regulating hematopoietic stem and progenitor cells[J];World Journal of Orthopedics;2013年04期
3 黄晓斌;孙元明;李雨民;杨福军;;破骨细胞分化成熟因子及其信号转导通路[J];中国骨质疏松杂志;2007年11期
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