The Effect Of Osteoblasts In G-CSF-Induced Hematopoietic Stem Cell Mobilization

Objective:Our purpose is to explore effects of hypoxia on osteoblasts’ proliferation and activity.Methods:Specimens from C57BL/6 mice injected with G-CSF at different time points are determined by flow cytometry, bone marrow immunohistochemistry, enzyme-labeled immunosorbent assay (ELISA). After osteoblasts are treated by G-CSF and cobalt chloride, we investigate the effects of hypoxic on osteoblasts’proliferation bycell count kits-8(CCK-8), osteoprotegerin (OPG) and ligand of receptor activator of NF-kB (RANKL) mRNA are detected after G-CSF and cobalt chloride treatment of osteoblasts by real-time fluorescence quantitative PCR, Westernblot is performed to detect the statusof OPG, RANKL and Hypoxia inducible factor-la (HIF-la).Results:① The number of LSK cells in peripheral blood significantly increased during mobilization, the proportion of LSK cells in peripheral blood on day0, day3 and day5 are 0.04±0.01%,0.10±0.02%,0.61±0.05%, respectively (P<0.05). ② The morphology of osteoblasts changes fromoval form to flat form during HSC mobilization. The number of osteoblasts decrease during mobilization, the number of osteoblasts in the trabecula of mouses on day0, day3 and day5 were 40.0±3.1 OB.N/B.s,23.0±2.5 OB.N/B.s and 10.0±1.7 OB.N/B.s, respectively (P<0.05). ③ The osteoblasts’activity decrease during mobilization, OCN protein levels on day0, day3 and day5 were 23.21±2.17ng/ml,9.46±1.66ng/ml,9.93±2.12ng/ml, respectively (P<0.05). ④ The growth rate of osteoblasts are treated by cobalt chloride 4h,24h,48h, 72h,96h,144h are 0.487±0.018,0.830±0.040,1.482±0.034,1.736±0.047, 2.141±0.052,2.515±0.049. The growth rate of control osteoblasts are 0.614±0.019,0.988±0.025,1.628±0.049,2.000±0.038,2.363±0.040, 2.741±0.075, The growth rate of osteoblasts are treated by G-CSF 4h,24h,48h, 72h,96h,144h are 0.559±0.038,0.992±0.020,1.619±0.073,1.920±0.089, 2.412±0.088,2.558±0.116. The growth rate of osteoblasts are treated by cobalt chloride are lower than control (P<0.05). There is no significant difference between the growth rate of osteoblasts are treated by G-CSF and control (P> 0.05). ⑤ The relative expression level of OPG Osteoblast after cobalt chloride stimulates islower than the control. The relative expression level of OPG the experimental group (osteoblasts are treatded by 100uM cobalt chloride 48 hours) and control group are 0.71±0.12、1.30±0.15, espectively (P<0.05). ⑥ The relative expression level of RANKL osteoblast after cobalt chloride stimulates is lower than the control, The relative expression level of RANKL in the experimental group (osteoblasts are treatded by 100uM cobalt chloride 48hours) and control group are 1.06±0.03 and 0.84±0.02, respectively (P<0.05). ⑦ The protein expression of osteoblasts’OPG and RNAKL decrease and hypoxia inducible factor-1 alpha increase after Osteoblasts are treated cobalt chloride.Conclusion: ① The decreases of osteoblast number and activity during G-CSF-induced HSC mobilization. ② Bone marrow low oxygen environment intensified may lead to the decrease of osteoblast number and activity during G-CSF induces HSC mobilization. ③ Induced by low oxygen inhibition of osteoblasts and HSC mobilization is probably one of the important mechanisms of induced HSC g-csf mobilization.