| Objective:To reveal the migratory capacity of bone marrow mesenchymal stem cells after incubation with erythropoietin.Methods:Healthy 6-8 weeks old female Japanese Large-eared White rabbits were used to isolate and expand rabbit BMMSCs by whole bone marrow culture method.In vitro induction of lipogenic osteogenic differentiation,CD29,CD44 and CD45 expression by flowmetry was performed at generation.P4 BMMSCs were incubated with different concentrations of EPO(0,100,200,300,1000 IU/ml)for 24 h and then assayed for proliferation using CCK-8.The groups were set,A: 0IU/ml EPO incubation group;B: 100IU/ml EPO incubation group;C:100IU/ml EPO+AMD3100 incubation group;D: AMD3100 incubation group alone.The effect of EPO on the migratory ability of BMMSCs was assessed using a scratch assay to detect the healed area of the scratch in each group after 24 h incubation.The migratory capacity of bone marrow mesenchymal stem cells after incubation with erythropoietin was assessed by detecting the number of cells crossing the membrane after 24 h incubation in each group using Transwell chambers.Total RNA and protein were extracted from each group of cells after 48 h incubation,and the m RNA and protein expression levels of SDF-1 and CXCR4 were detected using q RT-PCR and Western blot methods.Results:1.The cells were morphologically shuttle-shaped with swirling growth after passaged culture.High expression of CD29(98.6%)and CD44(98.9%)and low expression of CD45(0.97%)were detected by flow cytometry.In vitro with lipogenic osteogenic differentiation ability.2.0,100,200,300,1000 IU/ml EPO incubation groups with 24 h and 48 h OD450 values of 1.283±0.036,1.374±0.022,1.344±0.025,1.335±0.027,1.182±0.018;1.727± 0.041,1.827±0.029,1.789±0.033,1.737±0.029,1.574±0.027.Reflecting that in a certain concentration range,the proliferation of BMMSCs was concentration dependent on erythropoietin,with the most significant effect at a concentration of about 100 IU/ml(P<0.05),and a concentration of 1000 IU/ml inhibited the the proliferation of BMMSCs.3.After 24 h of cell scratching experiments in groups A,B,C and D,the healing rate of group B was statistically different higher than group A(P < 0.05),and that in group D was statistically different lower than group A(P < 0.05).4.The results of cell Transwell experiments in groups A,B,C and D.The numbers of cell migration in each group were 72±5.54,109±5.54,52.8±5.70,and 30.2±3.86.The group B number of cells spanning the membrane was statistically different higher than group A(P <0.05)and group D had statistically different less cell migration than control group A(P<0.05),group C had significantly lower cell migration compared with group B(P<0.05).The migration capability of BMMSCs was upgraded by adding EPO,and AMD3100 could inhibit this effect.5.SDF-1 m RNA expression was distinctively increased in groups B and C as opposed to the control group(P < 0.05),and in group D the SDF-1 m RNA expression was lower than group C(P < 0.05).CXCR4 m RNA volume of expression was distinctively increased in group B as opposed to the control group(P < 0.05).The CXCR4 m RNA volume of expression in group D was distinctively decreased as opposed to the control group(P < 0.05),CXCR4 m RNA volume of expression in group C was distinctively decreased as opposed to group B(P < 0.05),and CXCR4 m RNA volume of expression in group D was lower than that in group C(P < 0.05).6.SDF-1 protein volume of expression in groups B and C was significantly increased as opposed to the control group(P < 0.05),and SDF-1 protein expression was significantly decreased in group D compared with group C(P < 0.05),CXCR4 protein expression was distinctively increased in group B as opposed to the control group(P < 0.05),CXCR4 protein volume of expression was lower in group D as opposed to the control group(P < 0.05),and CXCR4 protein expression was distinctively decreased in group C as opposed to group B(P <0.05).Conclusion:EPO in a certain concentration range can promote the proliferation of BMMSCs,and EPO affects the migration of BMMSCs by upregulating the m RNA and protein expression of SDF-1 and CXCR4 through regulating the SDF-1/CXCR4 axis. |
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