| Objective:Bone Mesenchymal Stem Cells(BMSCs)were transfected with lentivirus to up-regulate the gene expression level of Chemotaxis cytokine receptor-4(CXCR4)in vitro.Then,the CXCR4 blocker AMD3100 and phosphoinositide 3-kinase(P13K)signaling blocker LY294002 were given to observe the changes in cell migration and apoptosis after the treatment of intervention factors,and to explore the effect of SDF-1/CXCR4 axis and downstream pathway PI3K/Akt on the migration function of BMSCs in vitro.Methods:Rat bone marrow mesenchymal stem cells were cultured in vitro,and the third-generation cells with good growth status were transfected with lentivirus carrying rat CXCR4 gene.Puromycin was used to screen out the cells with stable expression of rat CXCR4 gene,namely CXCR4-BMSCs.CCK8 proliferation assay was conducted to explore the optimal concentration of AMD3100 and LY294002 acting on BMSCs.The experiment was divided into four groups:I.BMSC group;II.CXCR4-BMSC group;?.AMD3100 group(CXCR4-BMSC+AMD3100 group);IV.LY294002 group(CXCR4-BMSC+LY294002 group).Each group was treated with 100ng/mlSDF-1,CCK8 kit was used to detect the cell proliferation activity of each group,and TUNEL assay was used to detect the cell apoptosis of each group.DMEM-F12 medium containing 100ng/ml SDF-1 was added to the subplate chamber of Transwell,and 4 groups of cells(1×104cells per well)were inoculated in the upper chamber.Drugs were added,and the cells were stained and counted after 12h to analyze the migration ability of cells in each group in vitro.mRNA and protein levels of CXCR4,PI3K and Akt were detected by RT-qPCR and Western blotting.Results:1.CCK8 proliferation experiments showed that the migration concentrations of AMD3100 and LY294002 were lOug/ml and 15umol/L,respectively.2.The proliferation of CXCR4-BMSCs was enhanced in the CXCR4-BMSCs group,and the proliferation of CXCR4-BMSCs was inhibited by AMD3100 and LY294002.3.TUNEL assay showed that the apoptosis rate in CXCR4-BMSC group was lower than that in BMSC group.Compared with the CXCR4-BMSC group,the apoptosis rates of AMD3100 group and LY294002 group were increased.4.Transwell assay showed that the transfected cells in BMSC group,CXCR4-BMSC group,AMD3100 group and LY294002 group were 49±5.6,78±11.2,65±10.3,60±9.5,respectively.Compared with BMSC group,CXCR4-BMSC group had stronger cell migration ability,and the difference was statistically significant(P<0.05).Compared with CXCR4-BMSC group,cell migration ability of AMD3100 group and LY294002 group was decreased,and the difference was statistically significant(P<0.05).5.RT-qPCR results showed that the mRNA levels of CXCR4,PI3K and Akt in the CXCR4-BMSC group were significantly higher than those in the BMSC group(P<0.05).Compared with the CXCR4-BMSC group,the mRNA expression levels of CXCR4,PI3K and Akt in the AMD3100 group were decreased,and the difference was statistically significant(P<0.05).mRNA expression levels of PI3K and Akt were decreased in LY294002 group,and the difference was statistically significant(P<0.05).6.Western blotting results showed that the protein expression levels of CXCR4,PI3K,Akt and p-akt in the CXCR4-BMSC group were higher than those in the BMSC group,with statistically significant differences(p<0.05).Compared with the CXCR4-BMSC group,the protein expression levels of CXCR4,PI3K,Akt and p-Akt in the AMD3100 group were decreased,and the difference was statistically significant(p<0.05).The protein expression levels of PI3K,Akt and p-Akt were decreased in LY294002 group,and the differences were statistically significant(p<0.05).Conclusion:1.The up-regulation of CXCR4 gene can promote the migration of BMSCs in vitro.2.The enhanced migration ability of BMSCs was consistent with the expression levels of CXCR4,PI3K and Akt,while AMD3100 and LY29002 can block this effect.3.SDF-1/CXCR4 axis and downstream PI3K/Akt signaling pathway are important signaling pathways promoting BMSCs migration in vitro. |
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