【摘要】：目的：粒細(xì)胞集落刺激因子誘導(dǎo)造血干細(xì)胞動(dòng)員過(guò)程中對(duì)成骨細(xì)胞的影響方法：本研究以C57BL野生型小鼠和人成骨細(xì)胞為研究對(duì)象,予C57BL野生型小鼠注射粒細(xì)胞集落刺激因子,應(yīng)用流式細(xì)胞儀、骨髓免疫組化、ELISA法檢測(cè)粒細(xì)胞集落刺激因子誘導(dǎo)造血干細(xì)胞動(dòng)員前后不同時(shí)點(diǎn)的小鼠外周血和骨髓標(biāo)本,觀察骨髓成骨細(xì)胞在粒細(xì)胞集落刺激因子誘導(dǎo)造血干細(xì)胞動(dòng)員前后的變化。粒細(xì)胞集落刺激因子及低氧模擬劑氯化鈷(CoCl2)處理人成骨細(xì)胞,應(yīng)用細(xì)胞計(jì)數(shù)試劑盒檢測(cè)人成骨細(xì)胞的增殖,以明確低氧對(duì)人成骨細(xì)胞增殖的影響。熒光定量PCR檢測(cè)經(jīng)粒細(xì)胞集落刺激因子和CoCl2處理前后人成骨細(xì)胞骨保護(hù)素基因和破骨細(xì)胞分化因子基因相對(duì)表達(dá)量的水平。westernblot檢測(cè)經(jīng)粒細(xì)胞集落刺激因子和CoCl2處理前后人成骨細(xì)胞骨保護(hù)素、破骨細(xì)胞分化因子和低氧誘導(dǎo)因子-1α的蛋白表達(dá)水平,明觀察低氧對(duì)人成骨細(xì)胞活性的影響。結(jié)果：①粒細(xì)胞集落刺激因子誘導(dǎo)動(dòng)員后小鼠外周血Lin-Scal+cKit+細(xì)胞占有核細(xì)胞的比例中day5顯著高于day0、day3 (0.61±0.05% VS 0.04±0.01%、0.10±0.02%, P0.05)。②粒細(xì)胞集落刺激因子誘導(dǎo)動(dòng)員過(guò)程中骨髓成骨細(xì)胞形態(tài)發(fā)生改變,形態(tài)由原來(lái)卵圓形變?yōu)楸馄叫�、梭形。粒�?xì)胞集落刺激因子誘導(dǎo)動(dòng)員后骨髓成骨細(xì)胞計(jì)數(shù)day5顯著低于day0、day3 (10.0±1.7VS 40.0±3.、23.0±2.5, OB.N/B.s, P0.05)。③粒細(xì)胞集落刺激因子誘導(dǎo)動(dòng)員后外周血OCN蛋白表達(dá)水平day 3和day5顯著低于dayO (9.93±2.12ng/ml、9.46±1.66ng/ml VS 23.21±2.17ng/ml, P0.05) ④CoCl2處理人成骨細(xì)胞4h、24h、48h、72h、96h、144h的增殖率分別為0.487±0.018、0.830±0.040、1.482±0.034、1.736±0.047、2.141±0.052、2.515±0.049,對(duì)照組人成骨細(xì)胞相應(yīng)時(shí)段的增殖率分別為0.614±0.019、0.988±0.025、1.628±0.049、2.000±0.038、2.363±0.040、2.741±0.075,粒細(xì)胞集落刺激因子處理人成骨細(xì)胞相應(yīng)時(shí)段的增殖率分別為0.559±0.038、0.992±0.020、1.619±0.073、1.920±0.089、2.412±0.088、2.558±0.116,經(jīng)COC12處理各時(shí)間段人成骨細(xì)胞的增殖率均顯著低于對(duì)照組(P0.05),經(jīng)粒細(xì)胞集落刺激因子處理各時(shí)間段人成骨細(xì)胞的增殖率與對(duì)照組差別均無(wú)顯著性意義(P0.05)。⑤實(shí)驗(yàn)組(100uM CoCl2培養(yǎng)48h)人成骨細(xì)胞骨保護(hù)素基因的相對(duì)表達(dá)量顯著低于對(duì)照組(0.71±0.12 VS 1.30±0.15,P0.05)。⑥實(shí)驗(yàn)組(100uM CoCl2培養(yǎng)48h)人成骨細(xì)胞破骨細(xì)胞分化因子基因的相對(duì)表達(dá)量顯著低于對(duì)照組(0.84±0.02 VS 1.06±0.03,P0.05)。⑦人成骨細(xì)胞經(jīng)CoCI2處理后,骨保護(hù)素和破骨細(xì)胞分化因子蛋白表達(dá)水平下調(diào),低氧誘導(dǎo)因子-1α蛋白表達(dá)水平顯著上調(diào)。結(jié)論：①粒細(xì)胞集落刺激因子誘導(dǎo)造血干細(xì)胞動(dòng)員過(guò)程中成骨細(xì)胞數(shù)量和活性顯著下降。②低氧可能是粒細(xì)胞集落刺激因子誘導(dǎo)造血干細(xì)胞動(dòng)員過(guò)程中成骨細(xì)胞數(shù)量和活性顯著下降的原因之一。③通過(guò)低氧抑制成骨細(xì)胞從而誘導(dǎo)HSC動(dòng)員可能是G-CSF誘導(dǎo)HSC動(dòng)員的重要機(jī)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 偽 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 鹵0.05% VS 0.04 鹵0.01) and 0.10 鹵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 鹵1.7VS 40.0 鹵3.30 鹵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 鹵2.12 ng / ml 9.46 鹵1.66ng/ml VS 23.21 鹵2.17 ng / ml, P0.05). 鈶oCl2澶勭悊浜烘垚楠ㄧ粏鑳，

本文編號(hào)：2201929