| Objective1)To explore the effect of exercise on urine-derived stem cells;2)To explore the effect of urine-derived stem cell exosomes with or without exercise intervention on anterior cruciate ligament reconstruction in vivo;3)To explore the effects and mechanism of urinary stem cell exosomes and exercise intervention on the function of vascular endothelial cells;Method(1)The urine of volunteers before and after exercise was collected to culture urine derived stem cells.Human urine derived stem cells were extracted by centrifugation and adherent methods.Urine derived stem cells identities and their differentiation potential were verified by lineage differentiation assays and stem cell surface antigens.The effects of exercise on the urinary stem cells were assessed by colony formation assays.(2)To explore the effect of urinary stem cell exosomes and exercise on anterior cruciate ligament reconstruction,rabbits were divided into three groups according to the operations: control group,USCs exosome group and USCs exosome with exercise group.There were 16 rabbits in each group,rabbits were sacrificed for histological staining,electron microscope scanning,PCR,and mechanical test.Eight rabbits were sacrificed in each group at 3 and 6 weeks,respectively.Each rabbit randomly selected one knee for anterior cruciate ligament reconstruction.The animal model of anterior cruciate ligament injury was established by cutting the anterior cruciate ligament at the insertion point.During the operation,the autologous semitendinosus tendon was taken for anterior cruciate ligament reconstruction.(3)Urine derived stem cell exosomes with /without exercise intervention were co cultured with human umbilical vein endothelial cells(HUVEC),and their influence on cell activity and angiogenic function were observed.The role of mir-210 in HUVEC cell activity was detected by specifically inhibit mir-210.Result(1)Colony formation Unit result showed that the number of colonies for urine derived stem cells in the group with exercise intervention was significantly larger;The morphology of urine derived stem cells changed with passaging,from polygon to spindle;Three lineage differentiation experiment showed that both groups of stem cells could be successfully induced into osteocyte,adipocyte and chondrocyte lineages.Urine derived stem cells markers CD29,CD90,CD73 and CD44 were positive in both groups,while hematopoietic stem cell markers CD34 and CD45 were negative in both groups;(2)The exosomes of the two groups were successfully extracted by ultra-high speed centrifugation from cell culture medium.Electron microscope showed that the exosomes in both groups display double-layer membrane structure with a diameter of about 100 nm;Particle size analysis showed that the concentration of stem cell exosomes in the exercise intervention group was higher than that in the control group;The exosomes of both groups expressed exosome surface antigens CD9 and TSG101;At 4 weeks after operation,A large number of cell infiltration were observed in the grafts in control surgery group.The collagen is disorganized in control group.Polarized light microscope showed that the gray scale value of tissue in control group was significantly lower than that in the SPT USCS exo group.At 8 weeks after operation,the collagen arrangement in the control group(CTL)was still chaotic.In the USCS exo group and SPT USCS exo group,the collagen was arranged orderly,and the cells were evenly distributed between the collagen fibers.Electron microscope showed that the collagen fibers of normal semitendinosus were uniform.The number and diameter of collagen fibers has changed after surgery,and a large number of collagen fibers with small diameter were visible.At 4 weeks after operation,the number of collagen fibers and fiber diameter in SPT-USCS-exo group and USCS-exo group were closest to those in normal semitendinosus muscle,while the number of collagen fibers in Ctl group was significantly higher than that in other groups,and the fiber diameter was also significantly smaller than that in other groups.At 8weeks after operation,the number of collagen fibers in Ctl group was significantly higher than that in USCS-exo group,and the diameterof collagen fibers was smaller than that in other groups.The mechanical results showed no dignificant difference between each group at 4weeks after surgery.At 8 weeks after surgery,the maximum failure load and stiffness were highest in SPT-USCS-exo group,followed by USCS-exo group and control operation group.The difference between the latter two groups was not significant.(3)After 48 hours of co culture,the proliferation,migration and angiogenesis potential of HUVEC cells in SPT-USCS-exo group were significantly higher than those in USCS-exo group;Angiogenesis related gene expression of vascular endothelial cells in SPT-USCS-exo group were significantly higher than those in USCS-exo group and control group.After blocking the effect of mir-210 with inhibitors,the angiogenic effect were decreased.Conclusion(1)Exercise can enhance the activity and potential of urine derived stem cells.(2)The exosome of urinary stem cells can promote the vascularization of anterior cruciate ligament reconstruction in vivo while exercise intervention could reinforce it.(3)Urinary stem cell exosomes and exercise intervention can promote the activity and angiogenesis of vascular endothelial cells in vitro.This effect is achieved by increasing the cargo of mir-210 in exosomes. |
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