粒细胞集落刺激因子诱导造血干细胞动员过程中对成骨细胞的影响
发布时间：2018-08-24 20:20
【摘要】：目的：粒细胞集落刺激因子诱导造血干细胞动员过程中对成骨细胞的影响方法：本研究以C57BL野生型小鼠和人成骨细胞为研究对象,予C57BL野生型小鼠注射粒细胞集落刺激因子,应用流式细胞仪、骨髓免疫组化、ELISA法检测粒细胞集落刺激因子诱导造血干细胞动员前后不同时点的小鼠外周血和骨髓标本,观察骨髓成骨细胞在粒细胞集落刺激因子诱导造血干细胞动员前后的变化。粒细胞集落刺激因子及低氧模拟剂氯化钴(CoCl2)处理人成骨细胞,应用细胞计数试剂盒检测人成骨细胞的增殖,以明确低氧对人成骨细胞增殖的影响。荧光定量PCR检测经粒细胞集落刺激因子和CoCl2处理前后人成骨细胞骨保护素基因和破骨细胞分化因子基因相对表达量的水平。westernblot检测经粒细胞集落刺激因子和CoCl2处理前后人成骨细胞骨保护素、破骨细胞分化因子和低氧诱导因子-1α的蛋白表达水平,明观察低氧对人成骨细胞活性的影响。结果：①粒细胞集落刺激因子诱导动员后小鼠外周血Lin-Scal+cKit+细胞占有核细胞的比例中day5显著高于day0、day3 (0.61±0.05% VS 0.04±0.01%、0.10±0.02%, P0.05)。②粒细胞集落刺激因子诱导动员过程中骨髓成骨细胞形态发生改变,形态由原来卵圆形变为扁平形、梭形。粒细胞集落刺激因子诱导动员后骨髓成骨细胞计数day5显著低于day0、day3 (10.0±1.7VS 40.0±3.、23.0±2.5, OB.N/B.s, P0.05)。③粒细胞集落刺激因子诱导动员后外周血OCN蛋白表达水平day 3和day5显著低于dayO (9.93±2.12ng/ml、9.46±1.66ng/ml VS 23.21±2.17ng/ml, P0.05) ④CoCl2处理人成骨细胞4h、24h、48h、72h、96h、144h的增殖率分别为0.487±0.018、0.830±0.040、1.482±0.034、1.736±0.047、2.141±0.052、2.515±0.049,对照组人成骨细胞相应时段的增殖率分别为0.614±0.019、0.988±0.025、1.628±0.049、2.000±0.038、2.363±0.040、2.741±0.075,粒细胞集落刺激因子处理人成骨细胞相应时段的增殖率分别为0.559±0.038、0.992±0.020、1.619±0.073、1.920±0.089、2.412±0.088、2.558±0.116,经COC12处理各时间段人成骨细胞的增殖率均显著低于对照组(P0.05),经粒细胞集落刺激因子处理各时间段人成骨细胞的增殖率与对照组差别均无显著性意义(P0.05)。⑤实验组(100uM CoCl2培养48h)人成骨细胞骨保护素基因的相对表达量显著低于对照组(0.71±0.12 VS 1.30±0.15,P0.05)。⑥实验组(100uM CoCl2培养48h)人成骨细胞破骨细胞分化因子基因的相对表达量显著低于对照组(0.84±0.02 VS 1.06±0.03,P0.05)。⑦人成骨细胞经CoCI2处理后,骨保护素和破骨细胞分化因子蛋白表达水平下调,低氧诱导因子-1α蛋白表达水平显著上调。结论：①粒细胞集落刺激因子诱导造血干细胞动员过程中成骨细胞数量和活性显著下降。②低氧可能是粒细胞集落刺激因子诱导造血干细胞动员过程中成骨细胞数量和活性显著下降的原因之一。③通过低氧抑制成骨细胞从而诱导HSC动员可能是G-CSF诱导HSC动员的重要机制之一。
[Abstract]:Objective: to investigate the effect of granulocyte colony stimulating factor (GSCF) on osteoblasts in the process of hematopoietic stem cell mobilization. Methods: in this study, C57BL wild-type mice were injected with granulocyte colony stimulating factor (GCSF). The peripheral blood and bone marrow samples of mice before and after hematopoietic stem cell mobilization induced by granulocyte colony stimulating factor were detected by flow cytometry and Elisa. To observe the changes of bone marrow osteoblasts before and after granulocyte colony stimulating factor induced hematopoietic stem cell mobilization. Human osteoblasts were treated with granulocyte colony stimulating factor (GCSF) and hypoxia-mimic cobalt chloride (CoCl2). The proliferation of human osteoblasts was detected by cell count kit in order to clarify the effect of hypoxia on the proliferation of human osteoblasts. Fluorescence quantitative PCR detection of relative expression of osteoprotegerin gene and osteoclast differentiation factor gene in human osteoblasts treated with granulocyte colony stimulating factor and CoCl2. Western blot detection of granulocyte colony stimulating factor and CoCl2 treatment Osteoprotegerin of human osteoblast before and after, The protein expression level of osteoclast differentiation factor and hypoxia inducible factor 1 伪 was observed to observe the effect of hypoxia on the activity of human osteoblasts. Results the percentage of Lin-Scal cKit cells occupying nuclear cells in peripheral blood of mice induced by 1: 1 granulocyte colony-stimulating factor was significantly higher than that of day0,day3 (0.61 卤0.05% VS 0.04 卤0.01) and 0.10 卤0.02 (P0.05). The morphology of bone marrow osteoblasts was changed during the mobilization induced by the granulocyte colony stimulating factor. The shape changed from oval to flat, fusiform. The count of bone marrow osteoblasts (day5) of granulocyte colony-stimulating factor induced mobilization was significantly lower than that of day0,day3 (10.0 卤1.7VS 40.0 卤3.30 卤2.5, OB.N/B.s, P0.05). The expression levels of day _ 3 and day5 in peripheral blood were significantly lower than dayO (9.93 卤2.12 ng / ml 9.46 卤1.66ng/ml VS 23.21 卤2.17 ng / ml, P0.05). 鈶oCl2澶勭悊浜烘垚楠ㄧ粏鑳，
本文编号：2201929
[Abstract]:Objective: to investigate the effect of granulocyte colony stimulating factor (GSCF) on osteoblasts in the process of hematopoietic stem cell mobilization. Methods: in this study, C57BL wild-type mice were injected with granulocyte colony stimulating factor (GCSF). The peripheral blood and bone marrow samples of mice before and after hematopoietic stem cell mobilization induced by granulocyte colony stimulating factor were detected by flow cytometry and Elisa. To observe the changes of bone marrow osteoblasts before and after granulocyte colony stimulating factor induced hematopoietic stem cell mobilization. Human osteoblasts were treated with granulocyte colony stimulating factor (GCSF) and hypoxia-mimic cobalt chloride (CoCl2). The proliferation of human osteoblasts was detected by cell count kit in order to clarify the effect of hypoxia on the proliferation of human osteoblasts. Fluorescence quantitative PCR detection of relative expression of osteoprotegerin gene and osteoclast differentiation factor gene in human osteoblasts treated with granulocyte colony stimulating factor and CoCl2. Western blot detection of granulocyte colony stimulating factor and CoCl2 treatment Osteoprotegerin of human osteoblast before and after, The protein expression level of osteoclast differentiation factor and hypoxia inducible factor 1 伪 was observed to observe the effect of hypoxia on the activity of human osteoblasts. Results the percentage of Lin-Scal cKit cells occupying nuclear cells in peripheral blood of mice induced by 1: 1 granulocyte colony-stimulating factor was significantly higher than that of day0,day3 (0.61 卤0.05% VS 0.04 卤0.01) and 0.10 卤0.02 (P0.05). The morphology of bone marrow osteoblasts was changed during the mobilization induced by the granulocyte colony stimulating factor. The shape changed from oval to flat, fusiform. The count of bone marrow osteoblasts (day5) of granulocyte colony-stimulating factor induced mobilization was significantly lower than that of day0,day3 (10.0 卤1.7VS 40.0 卤3.30 卤2.5, OB.N/B.s, P0.05). The expression levels of day _ 3 and day5 in peripheral blood were significantly lower than dayO (9.93 卤2.12 ng / ml 9.46 卤1.66ng/ml VS 23.21 卤2.17 ng / ml, P0.05). 鈶oCl2澶勭悊浜烘垚楠ㄧ粏鑳，
本文编号：2201929
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