| ObjectivesBone marrow mesenchymal stem cell(BMSCs)is a kind of adult pluripotent stem cell with high potential of multiple differentiation and high proliferation ability.BMSCs has been widely studied and used in the field of stem cell therapy so far.Homing is the key point related to the results of treatment with stem cells in damage tissue,which is the function of BMSCs on migrating and aggregating through blood circulation to the damaged tissue.There are two sources of BMSCs:in vivo and in vitro donors,but both the endogenous and exogenous BMSCs have very low homing rates in the target tissue.Hence,to improve the BMSCs'homing ability is a research focus in the field of stem cell therapy.Low intensity pulsed ultrasound(LIPUS)can enhance the proliferation and differentiation capacity of BMSCs in vitro,and promote the fracture healing of which mechanism is reported that LIPUS can be advisable in the healing of fracture by promote the mobilization and the supplement of BMSCs.Therefore,the aim of this research was to study the effect of LIPUS on migration and homing ability of BMSCs by in vivo and in vitro experiments and explore the mechanism through genome-wide expression profiling.The research will backed the application of LIPUS in stem cell therapy strongly.Methods1.To detect the well LIPUS intensity which can best improve the migration rate of BMSCs.BMSCs were divided into 4 groups:control group,30mW/cm~2 group,60mW/cm~2 group and 90mW/cm~2 group.The control group was given sham LIPUS exposure.The others were treated for20min by LIPUS whose intensity was 30mW/cm~2,60mW/cm~2 and90mW/cm~2 respectively.The effect of LIPUS on scratch healing was tested by scratch assay.The migration rate of BMSCs was evaluated by transwell migration assay.The expression of F-actin was stained by FITC and observed under fluorescence microscope.2.To explore the migration and homing rate of BMSCs in SD rats.Thirty female SD rats model that had caused femur bone defects in right limb were randomly divided into 2 groups:control group and LIPUS group(each group consist of 15 rats).Then the rats were injected with GFP-BMSCs which were treated by LIPUS(30mW/cm~2,20min/day,2d).The defects were detected by x-ray.After 2 weeks,the rats were sacrificed.The bone marrow was extracted and observed by HE staining.The homing rate of GFP-BMSCs in the target area was analyzed by fluorescence microscopy.3.To research the mechanism.The difference of gene expression between two groups was detected by genome-wide expression profiling,the differentially expressed genes were summarized,and then analyzed by gene annotation(GO).Four up-regulated genes were selected(Itga8,Ccl5,Tm7sf2,Tuba3b)to verify the expression levels by real-time fluorescence quantitative PCR.The temperature was monitored by a temperature inspection instrument during the LIPUS treatment.Results1.Compared to the control group,the scratch area of LIPUS groups were smaller at any detected times and the difference was statistically significant(P<0.05).The scratch area of 30mW/cm~2 group was the smallest among 4 groups at 24h(0.47±0.21mm~2)and 48h(0.19±0.10mm~2),the difference was statistically significant(P<0.05).In the transwell migration assay,the numbers of cells migrated through the transwell chamber in LIPUS groups were larger than the control group(P<0.05),and the migrated cell numbers of 30mW/cm~2 group was the most(213±34.76,P<0.05)among LIPUS groups.In the F-actin fluorescence staining assay,the cell morphology of the LIPUS groups was changed,and the cytoskeleton became narrow and elongated.The fluorescence intensity of the 30mW/cm~2 group was the largest(125.43±17.43,P<0.05).2.The bone defects model of SD rats was successfully established.Immediately after model establishment,the similar circular defects can be found in both two groups by x-ray imaging.After 2 weeks,the x-ray images showed that the defects in the LIPUS group were almost healed,while the control group had unhealed defects.HE staining showed that there were more osteoblasts in the bone marrow region of the LIPUS group.The fluorescence intensity of LIPUS group(12.06±0.23)was larger than the control group(38.2±0.29),the difference was statistically significant(P<0.05).3.The results of genome-wide expression profiling showed 237differentially expressed genes were screened out.Compared to the control group,39 genes were up regulated and 198 genes were down regulated in LIPUS group.33 genes were related to the G-protein coupled receptors activity in molecular function enrichment results(P<0.01);in biological process enrichment results,42 genes were associated with G-protein coupled receptor signaling(P<0.01);in molecular composition enrichment results,71 genes were related to cell membrane composition.The real-time fluorescence quantitative PCR results were consistent with expression profile sequencing,which meant the genome-wide expression profiling results in this study was effective and reliable.There was no significant difference in the temperature variation during the LIPUS treatment between control group and LIPUS group(P>0.05).ConclusionLIPUS can promote the migration ability of BMSCs in vitro and the well intensity was 30mW/cm~2.LIPUS pretreatment can be used for improving the homing rate of exogenous BMSCs,of which mechanism might be the G-protein coupled receptors on the membrane of BMSCs affected by mechanical effect of LIPUS.The LIPUS appears a tremendous potential in stem cell therapy. |
PDF Full Text Request