Effect And Mechanism Of MSCs Mobilization With Proline Hydroxylase Inhibitor DMOG In Vivo

Objective To investigate the characteristics of MSCs derived from peripheral blood(PB-MSCs)on the basis of MSCs mobilization with proline hydroxylase inhibitor DMOG in vivo,and explore the effect and mechanism of HIF-1 and its downstream SDF-1/CXCR4 and VEGF/VEGFR signaling pathway in MSCs mobilization mediating with DMOG.Methods(1)Exploration of MSCs mobilization with DMOG on rat:?DMale SD rats were randomly divided into two groups:DMOG group;Normal saline control group(NS group).DMOG group rats were treated with the dose of DMOG 40mg/kg 7 days by peritoneal injection,while the NS group rats were given the same volume of normal saline instead.(2)The characteristics of PB-MSCs in DMOG mobilization group:?PB-MSCs surface markers analysis by flow cytometry assay.?Neural,chondrogenic,osteogenic and adipogenic differentiation potential of PB-MSCs in vitro were assessed by morphological observation and specific identification.?The growth curve of PB-MSCs and MSCs derived from bone marrow(BM-MSCs)were detected by Cell Counting Kit-8.?PB-MSCs and BM-MSCs cell-cycle were examined by flow cytometry assay.?The migration ability of PB-MSCs and BM-MSCs were detected by Transwell assay.(3)Mechanism of HIF-1 and its downstream signaling pathway in mediating MSCs mobilization with DMOG:?Male SD rats were randomly divided into five groups:DMOG group,YC-1 group,AMD3100 group,SU5416 group,NS group.?The number of MSCs and the percentage of CD45-CD90+cells in rat peripheral blood and bone marrow was determined by CFU-F assay and flow cytometry,respectively.?The concentrations of SDF-la and VEGF both in PB and BM serum in each group were detected by ELISA.?Western blotting was used to test protein levels of HIF-1?,SDF-1? and VEGF in bone marrow cells.?Bone marrow microvessel quantity was detected by Immunohistochemistry.Results(1)Exploration of MSCs mobilization with DMOG on rat:?Compared with NS group,the number of CFU-Fs both in PB and BM increased in DMOG group(1.4±0.54 vs 2.6±0.54;9.2±0.83 vs 13.4 ± 1.14,P<0.05).?Compared with NS group,the percentage of CD45-CD90+cells increased both in PB and BM in DMOG group(2.94±0.06%vs 3.67 ±0.17%;6.37±0.23%vs 15.3 ±0.33%,P<0.05).(2)The characteristics of PB-MSCs in DMOG mobilization group:?The surface markers of PB-MSCs:PB-MSCs were negative for the hematopoietic lineage marker CD34(2.16±1.60%)and CD45(0.82±0.55%),and positive for CD90(98.66±0.77%).?PB-MSCs possessed a neural,chondrogenic,osteogenic and adipogenic differentiation potential.?PB-MSCs appeared to grow at a slow speed than BM-MSCs,the growth curve of PB-MSCs and BM-MSCs both exhibited a typical "S shaped curve",both of their proliferation possessed lag phase,exponential phase and stationary phase.?The result of cell-cycle detection:The proportion of PB-MSCs in S phase was significantly lower than BM-MSCs(10.15±0.64%vs 14±0.54%,P<0.05).?On the migration ability of SDF-1?,PB-MSCs is lower than BM-MSCs(108.6± 14.61 vs 133.4± 14.37/100×,P<0.05).(3)Mechanism of HIF-1 and its downstream signaling pathway in mediating MSCs mobilization with DMOG:?Compared with DMOG group,the number of CFU-Fs both in PB and BM decreased in YC-1?AMD3100?SU5416 group(2.6±0.54 vs 1.2±0.83?1.6±0.54?1.6±0.54;13.4±1.14 vs 9.8±0.83?10.4±1.14?9.6±1.14,P<0.05).?Compared with DMOG group,the percentage of CD45-CD90+cells decreased both in PB and BM in YC-1?AMD3100?SU5416 group(3.67±0.17%vs 2.52±0.05%?2.55±0.10%?2.48±0.07%;15.3±0.33%vs 12.6±0.31%?13.1±0.55%?11.6±0.53%,P<0.05).?Compared with DMOG group,the concentration and protein expression of HIF-1? decreased significantly in YC-1 group(P<0.05),the concentration and protein expression of SDF-1? decreased significantly in AMD3100 group(P<0.05),the concentration and protein expression of VEGF decreased significantly in SU5416 group(P<0.05).?Compared with NS group,the number of bone marrow microvessel increased in DMOG group(25±2.57 vs 38.7±4.96%,P<0.05);Compared with DMOG group,the number of bone marrow microvessel decreased in SU5416 group(38.7±4.96 vs 24.7±2.06%,P<0.05).Conclusions(1)DMOG has the effect of MSCs mobilization on rat,not only increased the number of MSCs both in PB and BM,but also improve the percentage of CD45-CD90+cells both in PB and BM.(2)The surface markers of PB-MSCs were negative for the hematopoietic lineage marker CD34 and CD45(0.82±0.55%)and positive for CD90,PB-MSCs possessed a neural,chondrogenic,osteogenic and adipogenic differentiation potential.Confirmed that the peripheral blood mononuclear cells cultured were MSCs in DMOG mobilization group;At the same time,PB-MSCs and BM-MSCs also have the proliferation and migration ability.(3)DMOG can increase the expression of HIF-1?,when inhibits the expression of HIF-1 by YC-1,the efficiency of mobilization decreases,the number of MSCs and the percentage of CD45-CD90+cells decreases both in PB and BM,suggesting that HIF-1? plays a key role in mediating MSCs mobilization with DMOG.(4)DMOG can increase the expression of SDF-1? and VEGF,when inhibits the pathway of SDF-1/CXCR4 by AMD3100 and inhibits the pathway of VEGF/VEGFR by SU5416,the efficiency of mobilization decreases,the number of MSCs and the percentage of CD45-CD90+cells decreases both in PB and BM,suggesting that HIF-1 downstream SDF-1/CXCR4 and VEGF/VEGFR signal pathway play a important role in mediating MSCs mobilization with DMOG.