Cross-talk Between BMP-2/Smad/Runx2/Osterix Axis And CXCL12/CXCR4 Axis In MSCs

PartⅠ Construction and Identification of the Recombinant Adenovirus Plasmid of c-x-c motif chemokine ligand 12Objective To construct the recombinant adenovirus expressing the c-x-c motif chemokine ligand 12(CXCL12).Methods:The CXCL12 gene fragment was amplified by PCR according to the c DNA of CXCL12,and then the fragment was cloned into p HBAd– MCMV-GFP.The recombinant plasmid was co-transfected into 293 T cells with packing plasmids for packaging and amplifying.Infection titer and rate were monitored by green fluorescent protein expression.The expression of CXCL12 were detected by ELISA and QPCR after Ad-CXCL12 infected MSCs.Results:The results of sequence scan,restriction endonuclease,and PCR confirmed that CXCL12 was cloned into the adenovirus vector successfully.The protein and m RNA levels of CXCL12 were increased significantly after Ad-CXCL12 infected MSCs.Conclusion:The recombined adenovirus p HBAd-MCMV-CXCL12-GFP was constructed successfully,and it can be used in the next.PartⅡ Effect of Runx2 on migration of mesenchymal stem cellsObjective To explore the role of runt-related transcription factor-2(Runx2)in the migration of mesenchymal stem cells(MSCs)in vitro.Methods MSCs were isolated from whole bone marrow of rabbits,and incubated with Ad-Runx2,Ad-GFP or AMD3100.By using Transwell test to observed the effects of Runx2 concentrations on MSCs migration.The expression of c-x-c motif chemokine ligand 12(CXCL12)/ Cys-X-Cys receptor 4(CXCR4)m RNA was evaluated by QPCR.The expression of CXCL12 protein levels was detected by ELISA.The expression of CXCR4 protein levels was detected by Western-blot.Results The transmigrated cell number was significantly up-regulated with the addition of Ad-Runx2(p<0.01).The transmigrated cell number was decreased by AMD3100(P<0.01).The protein and m RNA levels of CXCL12 and CXCR4 were increased after Ad-Runx2 infected MSCs for 72 h.The expression level of CXCL12 and CXCR4 were decreased after incubation with AMD3100(p<0.01).Conclusion These results indicated that Runx2 had a promotive effect on MSCs migration,and achieved through increasing expression of CXCL12 and CXCR4.PartⅢ Effects of altered CXCL12 on BMP2/Smad/Runx2/Osterix axis and osteogenic gene expressions during osteogenic differentiation of MSCsObjective To observe the effects of CXCL12/CXCR4 axis on signal axis BMP-2/Smad/Runx2/Osterix and osteogenic gene expressions during osteogenic differentiation of MSCs,to understand the intrinsic link between the migration signal axis and osteogenic differentiation signal axis,and to provide theoretical basis for further understanding osteogenic differentiation mechanism of MSCs.Methods p HBAd-MCMV-CXCL12-GFP vector(Ad-CXCL12)was constructed. Quantitative polymerase chain reaction(q PCR)and western blot assay were used to determine CXCL12 expression in Ad-CXCL12-transfected MSCs.MSCs were then treated with Ad-CXCL12 and AMD3100(CXCL12 inhibitor)so as to detect BMP-2/Smad /Runx2/ Osterix expression,BSP,OCN,OPN m RNA expressions,and ALP activity.Results(1)PCR and sequencing results confirmed that CXCL12 recombinant adenovirus vector was successfully constructed.q PCR and enzyme linked immunosorbent assay results showed that Ad-CXCL12 transfection promoted CXCL12 expression in MSCs.(2)At 72 hours,Runx2 and Osterix,Smad1/5/8 m RNA and protein expressions were significantly higher in the Ad-CXCL12 group than in the control group(P < 0.01).(3)At 1 week,ALP activity was significantly higher in the Ad-CXCL12 group than in the control group(P < 0.01).At 2 weeks,BSP m RNA expression was higher in the Ad-CXCL12 group than in the control group(P < 0.01).No significant difference in OCN and OPN m RNA expressions was determined between the Ad-CXCL12 and control groups(P > 0.05).(4)At 3 weeks,the staining of mineralized nodules did not demonstrate significant differences between Ad-CXCL12 group and other groups(P > 0.05).Conclusion The changes in migration signal axis CXCL12/CXCR4 affect BMP-2/Smad/Runx2/Osterix signal axis,BSP,OCN,and OPN m RNA expressions in the early stage of osteogenic differentiation of MSCs,thereby affecting the ability of osteogenic differentiation of MSCs.These effects of CXCL12/CXCR4 axis were not obvious in the middle and late stages of osteogenic differentiation of MSCs.