| ObjectsTo investigate the effect of SDF-1/CXCR4 axis on the migration and colonization of bone marrow mesenchymal stem cells into bone marrow in mice with bone marrow failure.Methods1.The fusion gene fragment of CXCR4 and Luciferase was transfected by lentivirus to make healthy human bone marrow-derived mesenchymal stem cells overexpress CXCR4.The cells were labeled CXCR4-BMSCs.At the same time,the cells were labeled Null-BMSCs as control group,which was transfected Luciferase gene fragment.2.The expression of CXCR4 in cells was detected by Q-PCR and flow cytometry.3.The biological properties of CXCR4-BMSCs were studied and identified:Osteogenic and adipogenic induction assay was used to detect the osteogenic and adipogenic ability of cells.The cell viablility was measured by CCK-8 assay.the cell apoptotic rate and cell cycle was analyzed by flow cytometry.4.The migration ability to SDF-1 of BMSCs cultured under hypoxia,BMSCs pretreated with Ligustrazine and CXCR4-BMSCs was compared in transwell system.5.The protein level of CXCR4 in CXCR4-BMSCs and normal BMSCs was detected by Western blot.6.The model of bone marrow failure in mice was established.The blood and bone marrow of the model mice were examined and analyzed by blood cell analysis and bone marrow smear.7.Different cell transfusion treatments were given to mice in each group:normal group(CXCR4-BMSCs infusion,2*106 cells per mouse);model group 1(PBS buffer infusion only),model group 2(Null-BMSCs infusion,2*106 cells per mouse);model group 3(CXCR4-BMSCs infusion,2*106 cells per mouse).8.The distribution of transfused cells in mice was traced by in IVIS Lumina ? system and the positive marker cells in bone marrow were detected by flow cytometry on the second day after cell infusion.9.The secretion of SDF-1 in peripheral blood and bone marrow in mice was measured by ELISA.Results1.Compared with Null-BMSCs,CXCR4-BMSCs overexpressed CXCR4.CXCR4-BMSCs have the ability of osteogenesis and adipogenesis.There was no significant difference between CXCR4-BMSCs and normal BMSCs in cell activity(106%±13%vs 99%±9%),apoptotic rate(1.212%±0.357%vs 1.186%±0.727%)and cell cycle.2.Compared with normal BMSCs,BMSCs cultured under hypoxia,BMSCs pretreated with Ligustrazine(100 p mol/L)had higher migration ability to SDF-1,and CXCR4-BMSCs had highest migration ability(163.4±20.452).3.The protein level of CXCR4 in CXCR4-BMSCs was higher than that in normal BMSCs.4.The hemogram and bone marrow of the model mice accorded with the level of bone marrow failure,and the model of bone marrow failure was successfully constructed.5.In vivo imaging showed that CXCR4-BMSCs were concentrated in the femur in bone marrow failure mice.Compared with other groups of mice,CXCR4-BMSCs had more intensive fluorescence signals.The results of flow cytometry showed that the percentage of positive labeled cells in bone marrow of model group 3 was higher(23.650+2.489%).The efficiency of homing cells with high expression of CXCR4 gene to bone marrow was improved.6.Compare with the SDF-1 level(222.963±14.908 pg/L)in bone marrow of normal mice,SDF-1 level(415.622±37.029 pg/L)of model mice were significantly increased.The level of SDF-1 in bone marrow was higher than that in peripheral blood(21.390±2.305 pg/L).Conclusion1.CXCR4-BMSCs transfected by lentiviruses overexpress CXCR4.There is no significant difference in cell biological properties between lentiviral transfected cells and normal BMSCs,and they have osteogenic and adipogenic abilities.2.After BMSCs were treated with Ligustrazine,the expression of CXCR4 protein increased and the migration ability increased.However,CXCR4-BMSCs had stronger migration ability because of the overexpression of CXCR4.3.In mice with bone marrow failure,the migration and colonization ability of CXCR4-BMSCs to bone marrow was significantly improved.The SDF-1 level in bone marrow failure mice was higher than that in normal mice.SDF?1/CXCR4 axis plays an important role in promoting BMSCs homing. |
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