The Effect Of G-CSF On The Expression Of Osteoblast And OCN、SDF-1、Ang1in Mice

Objective Peripheral blood stem cell transplantation (PBSCT) has been widelyused in the treatment of various malignant or non-malignant hematologicaldiseases and some genetic diseases. How to successfully mobilize enoughhematopoietic stem cells (HSC) into peripheral blood and collect them is thepremise of successful treatment with PBSCT. But, how to make themobilization more effectively and useful is still a hard knot in treatment withHSC. Although mechanism about the mobilization of stem cells is not yet fullyclear, many scholars acknowledged the change of bone marrow play a role inmobilization of stem cells. Osteoblasts are the main components of the HSCniches, they adjust the number of HSC by control the number of osteoblast inthe HSC niche, which resulted in the damage of HSC steady state. This studywas conducted to investigate the changes of osteocaicin (OCN) and osteoblastssecrete factors Ang1in the mobilization process, large doses of PTHinterference targeting osteoblasts, explore the effect and the molecularmechanisms about the change of endosteal osteoblasts in stem cell mobilizationprocess.Methods Specimens（peripheral blood and bone marrow） from C57BL/6miceinjected with G-CSF at different time points(0、3、5day) were determined by bone marrow immunohistochemistry, ELISA, and real-time fluorescencequantitative PCR. Observation changes of OCN and Ang1before and aftermobilization. Finally, flow cytometry was used to measure the change ofperipheral blood stem cell mobilization in mice, whose bone marrow osteoblastswere suppressed by large doses of PTH, to investigate the effect of osteoblastsand the relevant mechanism in the G-CSF-mediated HSC mobilization process.Results1. The use of stem cell mobilization and G-CSF can lead to a reduction in thenumber and activity of mouse bone cells: In the process of GCSF-inducedstem cell mobilization, the morphological changes of osteoblasts could beseen under HE staining, steady bone osteoblasts are oval and seriallydistribute along the bone cortex, but after effective mobilization, theirmorphology turned into fusiform and their quantity became sparse and less.The similar changes were consistent with the immunohistochemistry of OCN.Through counting found, the number of osteoblasts(OB.N/B.s) in eachbone surface of normal mouse respectively were:14.83士0.23(beforemobilization) and6.78士0.17(day5in mobilization)(p＜0.01). Osteoblastactivity was significantly decreased in mobilization day3and day5. Duringthe mobilization, OCN (osteoblast-specific gene) in day0, day3day5respectively were63.01±10.76、35.40±6.06and17.57±1.97. OCN relativeexpression level in day3and day5was significantly lower than thesteady-state （n=5，p＜0.01).2. The change of SDF-1、 Ang1in bone marrow after G-CSF inducedmobilization：Ang1gene relative expression on steady-state (day0), day3and day5respectively were4.28±1.30、2.28±0.39and1.27±0.34，day5wassignificantly lower than steady-state（n=5，p=0.015）. SDF-1gene relative expression on steady-state (day0), day3and day5respectively were4.41±1.56、1.37±0.54and0.96±0.28，day3and day5was significantly lowerthan steady-state（n=5，P＜0.01). Bone marrow samples ELISA test shown,Ang1protein respectively were11.89±1.62ng/m1、2.29±0.92ng/m1、0.62±0.17ng/ml in day0, dany3and day5. Steady-state was significantlydifferent from day3and day5（n=5，P＜0.01）.3. The change of OCN、Ang1proteins in the peripheral blood after G-CSFinduced mobilization: Peripheral blood OCN protein was significantdecreased during mobilization, day0, day3and day5respectively were21.13±3.14ng/m1,10.76±2.18ng/m1,10.32±1.78ng/ml， steady-state wassignificant different from day5（n=5，P＜0.01）. Peripheral blood Ang1protein in mobilization day0day3and day5respectively were2242.4±625.07ng/m1、1283.3±584.75ng/m1、966.6±299.06ng/ml，steady-state wassignificant different from day5（n=5，P=0.020）.4. The change of LSK cells after G-CSF combined PTH induced mobilization:Large doses of PTH suppression of osteoblast niches, percentage of theamount of mouse peripheral blood stem cells in mobilization day0, day3andday5were respectively increased from0.54±0.11%,2.20±0.34%and2.83±0.37%to1.70±o.46%,3.19±0.43%and4.32±0.49%（n=5,P<0.01).ConclusionIn the process of stem cell mobilization, a decline in the number andfunction of osteoblasts has led to the occurrence of mobilization. By inhibitingthe activity of osteoblasts may enhance mobilization efficiency.