脉冲电磁场对成骨细胞作用的研究
发布时间：2018-10-24 07:02
【摘要】：目的：一、研究脉冲电磁场(puslsed electromagnetic fields,PEMFs)对成骨细胞(Osteoblast, OB)的作用,探究PEMFs治疗骨质疏松的的作用机理。二、研究PEMFs对造血干细胞龛中的主要组成成分——ALCAM+Sca-1-成骨细胞的作用,探究PEMFs对造血干细胞归巢的作用。 方法：一、取新生SD大鼠头盖骨,通过骨组织块培养法分离、培养成骨细胞。取传代第三代细胞分为5组：1-70组、2-70组、3-70组、4-70组、对照组,其中前4组为作用组,分别接受1mT,70Hz；2mT,70Hz；3mT,70Hz；4mT,70Hz的PEMFs作用,对照组不接受PEMFs作用,同PEMFs组一致置于相同环境下、磁场作用距离外。按照分组剂量进行离体PEMFs作用(0.5h/d,连续作用4d),作用结束后,对各组分别进行细胞碱性磷酸酶(alkaline phosphatase, ALP)活性、核结合因子α1(core-binding factorα1, cbfα1/RUNX2)表达、体外成骨能力指标——矿化结节(mineralized nodules)形成能力检测。 二、24只C57/6小鼠随机分为两组：PEMF组和对照组,每组12只小鼠。PEMF组小鼠接受PEMFs作用,剂量为2mT,70Hz,1h/次,1次/d,对照组不接受PEMFs作用,同PEMFs组一致置于相同环境下、磁场作用距离外,1h次,1次/d。连续作用4w。作用结束后,两组分别取股骨、胫骨,冲出骨髓(bone marrow, BM)后,研磨至碎片,经胶原酶消化,收集细胞经淋巴细胞分离液分离,磁珠系统分选出CD31-CD45-TER119-细胞,再经流式细胞仪分选ALCAM+Sca-1-成骨细胞。分选出的ALCAM+Sca-1-成骨细胞经TRIzol法提取Total RNA,逆转录,基因芯片杂交,洗脱,检测,进行数据分析。 结果：一、经PEMFs作用后,与对照组相比,成骨细胞ALP活性降低,cbfα1表达增多,矿化结节数量显著增多。 二、经PEMFs作用后,PEMF组与对照组相比,ALCAM+Sca-1-.成骨细胞的基因上调表达成骨细胞功能相关基因,与造血干细胞归巢相关的细胞粘附分子、趋化分子基因,调节造血干细胞的细胞因子基因。结论：一、PEMFs作用成骨细胞可促进成骨细胞功能分化,增强成骨细胞的细胞外 基质矿化。 二、PEMFs作用促使造血干细胞龛中的主要组成成分——ALCAM+Sca-1-成骨细胞上调表达造血干细胞归巢相关基因,在基因水平上推断PEMFs有助于造血干细胞归巢重建造血功能。
[Abstract]:Objective: to investigate the effect of pulsed electromagnetic field (puslsed electromagnetic fields,PEMFs) on (Osteoblast, OB) of osteoblasts and explore the mechanism of PEMFs in the treatment of osteoporosis. Secondly, to study the effect of PEMFs on ALCAM Sca-1- osteoblast, the main component of hematopoietic stem cell niche, and to explore the effect of PEMFs on homing of hematopoietic stem cell. Methods: firstly, osteoblasts were isolated from the skull of newborn SD rats by bone mass culture. The third passage cells were divided into 5 groups: 1-70 group, 2-70 group, 3-70 group, 4-70 group, and control group. The first four groups were treated with 1mTTn70Hzn2mT70Hzn2mT70HzHzO3mTZN 4mTzTn70 Hz PEMFs, the control group was not treated with PEMFs, and was placed in the same environment as PEMFs group, the former group was treated in the same environment, and the control group was treated in the same environment as the PEMFs group. The magnetic field acts outside the range. PEMFs was treated in vitro (0.5 h / d for 4 days) according to the group dose. The alkaline phosphatase (alkaline phosphatase, ALP) activity and the expression of core-binding factor 伪 1 (cbf 伪 1/RUNX2) were observed in each group after the treatment. The ability of mineralized nodules to form (mineralized nodules) was measured as an index of osteogenic ability in vitro. Second, 24 C57 / 6 mice were randomly divided into two groups: PEMF group and control group, 12 mice in each group. The mice in PEMF group were treated with PEMFs at a dose of 2mTTn70Hz1, once a day. The control group was not treated with PEMFs, and was placed in the same environment as PEMFs group. Outside the magnetic field, 1 time, 1 time per day. Continuous action for 4 weeks. After the treatment, femur, tibia, bone marrow (bone marrow, BM) were washed out, then ground to pieces, digested by collagenase, collected cells were separated by lymphocytes, and CD31-CD45-TER119- cells were separated by magnetic beads system. ALCAM Sca-1- osteoblasts were separated by flow cytometry. The selected ALCAM Sca-1- osteoblasts were extracted Total RNA, reverse transcription, gene chip hybridization, elution, detection and data analysis by TRIzol. Results: first, compared with the control group, the ALP activity of osteoblasts decreased, the expression of cbf 伪 1 increased, and the number of mineralized nodules increased significantly after PEMFs treatment. Second, after treated with PEMFs, ALCAM Sca-1-. in PEMF group was higher than that in control group. The genes of osteoblasts up-regulate the expression of osteoblast function-related genes, the cell adhesion molecules and chemotactic molecules associated with homing of hematopoietic stem cells, and the cytokine genes of hematopoietic stem cells. Conclusion: firstly, PEMFs can promote the differentiation of osteoblasts and enhance the extracellular matrix mineralization of osteoblasts. Secondly, ALCAM Sca-1- osteoblast, the main component of hematopoietic stem cell niche, was induced by PEMFs to up-regulate the expression of homing related genes of hematopoietic stem cells. It was concluded that PEMFs could help hematopoietic stem cells to homing and reconstructing hematopoietic function at the gene level.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2290661
[Abstract]:Objective: to investigate the effect of pulsed electromagnetic field (puslsed electromagnetic fields,PEMFs) on (Osteoblast, OB) of osteoblasts and explore the mechanism of PEMFs in the treatment of osteoporosis. Secondly, to study the effect of PEMFs on ALCAM Sca-1- osteoblast, the main component of hematopoietic stem cell niche, and to explore the effect of PEMFs on homing of hematopoietic stem cell. Methods: firstly, osteoblasts were isolated from the skull of newborn SD rats by bone mass culture. The third passage cells were divided into 5 groups: 1-70 group, 2-70 group, 3-70 group, 4-70 group, and control group. The first four groups were treated with 1mTTn70Hzn2mT70Hzn2mT70HzHzO3mTZN 4mTzTn70 Hz PEMFs, the control group was not treated with PEMFs, and was placed in the same environment as PEMFs group, the former group was treated in the same environment, and the control group was treated in the same environment as the PEMFs group. The magnetic field acts outside the range. PEMFs was treated in vitro (0.5 h / d for 4 days) according to the group dose. The alkaline phosphatase (alkaline phosphatase, ALP) activity and the expression of core-binding factor 伪 1 (cbf 伪 1/RUNX2) were observed in each group after the treatment. The ability of mineralized nodules to form (mineralized nodules) was measured as an index of osteogenic ability in vitro. Second, 24 C57 / 6 mice were randomly divided into two groups: PEMF group and control group, 12 mice in each group. The mice in PEMF group were treated with PEMFs at a dose of 2mTTn70Hz1, once a day. The control group was not treated with PEMFs, and was placed in the same environment as PEMFs group. Outside the magnetic field, 1 time, 1 time per day. Continuous action for 4 weeks. After the treatment, femur, tibia, bone marrow (bone marrow, BM) were washed out, then ground to pieces, digested by collagenase, collected cells were separated by lymphocytes, and CD31-CD45-TER119- cells were separated by magnetic beads system. ALCAM Sca-1- osteoblasts were separated by flow cytometry. The selected ALCAM Sca-1- osteoblasts were extracted Total RNA, reverse transcription, gene chip hybridization, elution, detection and data analysis by TRIzol. Results: first, compared with the control group, the ALP activity of osteoblasts decreased, the expression of cbf 伪 1 increased, and the number of mineralized nodules increased significantly after PEMFs treatment. Second, after treated with PEMFs, ALCAM Sca-1-. in PEMF group was higher than that in control group. The genes of osteoblasts up-regulate the expression of osteoblast function-related genes, the cell adhesion molecules and chemotactic molecules associated with homing of hematopoietic stem cells, and the cytokine genes of hematopoietic stem cells. Conclusion: firstly, PEMFs can promote the differentiation of osteoblasts and enhance the extracellular matrix mineralization of osteoblasts. Secondly, ALCAM Sca-1- osteoblast, the main component of hematopoietic stem cell niche, was induced by PEMFs to up-regulate the expression of homing related genes of hematopoietic stem cells. It was concluded that PEMFs could help hematopoietic stem cells to homing and reconstructing hematopoietic function at the gene level.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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