Effects Of Endothelial Progenitor Cells Transplantation Combined-with GLP-1 Receptor Agonist Treatment On Hindlimb Ischemic Diabetic Rats In Microcirculation Angiogenesis

Objective:To study the effectivity and safety of endothelial progenitor cells(EPCs)transplantation and glucagon-like peptide 1(GLP-1)receptor agonist by intervention on hindlimb ischemia of diabetic rats.Method:1.To establish the acute hind limb ischemia on SD rats with diabetes mellitus.Diabetic SD rats with hind limb ischemia(n=80)were divided into 7 groups for transplantation.Group A1(n=10):EPCs transplantion and GLP-1 receptor agonist intervention.Group A0(n=10):EPCs transplantation.Group B1(n=12):bone marrow mononuclear cells transplantation and GLP-1 receptor agonist intervention.Group B0(n=12):bone marrow mononuclear cells transplantation.Group C1(n=12):normal saline and GLP-1 receptor agonist intervention.Group C2(n=12):normal saline and insulin intervention.Group C0(n=12):normal saline intervention.2.Diabetes was induced by streptozotocin in 24 SD male rats.Bone marrow mononuclear cells were isolated from these rats by density gradient centrifugation and cultured in fibronectin-coated dishes.The bone marrow-derived EPCs were labeled with CM-Dil before transplantation.Bone marrow mononuclear cells and EPCs were respectively transplanted by local multi-point injection to the affected limbs.Glucagon-like peptide 1(GLP-1)receptor and insulin were respectively hypodermical injected at different groups.3.The tissue samples of the muscle in the affected limbs was collected after all the rats were sacrificed in 28 days.The density of capillary was observed by Von Willebrand factor(vWF)related antigen immunochemical stain.The expression of vascular endothelial growth factor(VEGF)in the ischemic area was detected by ELISA assays.The expression of endothelial nitric oxide synthase(eNOS)was assessed by Western blot.4.After the experimental animals were sacrificed,sampled the affected limbs,contralateral limbs,hearts,eyes,lungs,and kidneys were observed for CM-Dil labeled cells.5.Monitor on tumor and acute immunological rejection after stem cells transplantation for all the experimental animals.Results:1.The levels of VEGF,eNOS and the densities of capillary in the ischemic area were significantly elevated in EPCs transplantation(group A1,A0)compared with controls(group C1,C0)(P<0.05).2.The levels of VEGF,eNOS and the densities of capillary in the ischemic area were significantly elevated in GLP-1 receptor agonist intervention combined with stem cells transplantation(group A1,B1)compared with stem cells transplantation(group A0,B0)(P<0.05).3.The levels of VEGF,eNOS and the densities of capillary in the ischemic area were significantly elevated in EPCs transplantation(group A1,A0)compared with bone marrow mononuclear cells transplantation(group B1,B0)(P<0.05).4.The-densities of capillary was significantly elevated in GLP-1 receptor agonist intervention(group C1)compared with controls(group C0)(P<0.05).No significant differences were observed between GLP-1 receptor agonist treated group(group Ci)and insulin treated group(group C2)on VEGF,eNOS levels and capillary density.Conclusion:? EPCs transplantation increases the levels of VEGF,eNOS and the densities of capillary in the ischemic area than bone marrow mononuclear cells transplantation.? GLP-1 receptor agonist treatment combined with EPCs transplantation further increases the level of VEGF,eNOS and the density of capillary in the ischemic area.