| Objective:Endothelial progenitor cells (EPCs) are precursor cells of angiogenesis, which can promote vascular endothelial repairing and angiogenesis. With experiments in vivo and in vitro studies high sugar environment inducing oxidative stress affect endothelial progenitor transplantation effect of the treatment of diabetes hind limbs ischemia, exploring the oxidative stress reaction endothelial progenitor cells WNT/beta-catenin signaling pathway change, leading to endothelial progenitor cell transplantation efficacy change possible mechanism.Methods In vitro experiment:put to death in the S-D rat dislocated neck method, collection of lower limb bone marrow mononuclear cells in the cavity, using Ficoll density gradient centrifugation and adherent culture method to separate and culture endothelial progenitor, using continuous morphological observation, identification of EPCs; Using molecular probe by the flow cytometry instrument detection (DCFH-DA) normal sugar concentration (N= 5.5 mol/L), high sugar content (HG= 30 mol/L) after processing the EPCs levels of oxidative stress. According to different methods can be divided into seven groups:N, HG, HG+DMSO (solvent XAV-939), DM+Licl (beta-catenin activator), HG+tempol (inhibitor of oxidatie stress), HG+H2O2, HG+ tempol+XAV-939 (beta- catenin inhibitors EPCs) after processing, EPCs adhesion of collagen crystal violet method were used to detect the cell number and transwell small number of cells migrating test chamber method, using Western blotting test the wnt/beta-catenin signaling pathways important related proteins wnt5a, T-beta-catenin (Total-beta-catenin), NP-beta-catenin (non phosphorylated-beta-catenin) and protein mediated adhesion function the situation of the expression of VCAM-1, In vivo experiment:chain urea with intraperitoneal injection of cephalosporins in diabetic rats model was established, the continuous monitoring of blood sugar,2 months after the success of the diabetic rats model into the group, operating microscope downward on the left side of the ligation of femoral artery and its branches from broken with diabetes unilateral hind limbs ischemia model was established, and compare the EPCs transplantation cases, not healthy rats and diabetic rats hind limbs ischemia in the model. Socket with health, hind limbs of rats bone marrow mononuclear cells by the method of separation, cultivate EPCs, postoperative 24 hours according to the quantity of 107/100 g move by tail vein injection into diabetic rats hind limbs ischemia model (DM+EPCs), in the preoperative and postoperative (0) d,7 d and 14 d and 28 d line continuous LDPI (laser doppler perfusion imager) check transplant to observe the efficacy of treatment. Is divided into three groups:healthy hind limbs ischemia model of rats by tail vein injection of saline solution (H+NS), diabetic hind limbs ischemia model of rats by tail vein injection of saline (DM+NS) and diabetic rats hind limbs ischemia model by tail vein injection of endothelial progenitor cells (DM+EPCs),28 days after peripheral blood MDA and antioxidant index SOD levels by spectrophotometric detection oxidative stress metabolites. Divided into seven groups:H+NS, DM+NS, DM+EPCs, DM+EPCs+ DMSO,DM+EPCs+Tempol, DM+EPCs+Licl and EPCs+Tempol+XAV 939, by the method of separation of EPCs, after PKH-26 tag tracer, according to the quantity of 107/100 g transplantation by tail vein injection into each hind limbs ischemia model of rats, the preoperative and postoperative (d) 0,7, 14,28 consecutive LDPI is checked, and 28 days after surgery in rats left hind leg and peripheral blood of the gastrocnemius muscle, using ELISA to detect serum wnt3a, wnt5a and wnt7a levels, and filter may play a role of regulating factor; Peripheral blood by spectrophotometric detection oxidative stress metabolites and antioxidant index MDA level; Via molecular probe DCFH-DA ROS and DHE in fluorescence microscope ischemia gastrocnemius oxidative stress level;Observed under fluorescence microscope lens ischemia gastrocnemius endothelial marker CD31 in blood vessel number and PKH-26 tracer migration, adhesion cells;Using Western blotting detection of ischemia gastrocnemius WNT/beta-catenin signaling pathways important related proteins wnt5a, T-beta-catenin (beta-catenin), NP-beta-catenin (not phosphorylation-beta-catenin) and protein mediated adhesion function expression of VCAM-1.Results:EPCs by special EGM-2 cultured with 2 d, EPCs as adherent cells cultured, assumes the circular, the little oval. observation can be found that "pebbles" cells in the sample, sample spiral growth.By laser confocal microscopy cells visible after dealing with the DiI ac-LDL EPCs in red fluorescence positive, combined with FITC EPCs after UEA-1 for yellow fluorescence, double staining positive cells to differentiate the EPCs, shows that the cultured cells of EPCs. Continue to develop to 7 d after the cell increases, into a clear oval or spindle grows fast;After 14 d, observation can be found that "pebbles" cells in the sample, sample spiral growth. By laser confocal microscopy cells visible after dealing with the DiI-ac-LDL EPCs in red fluorescence positive, combined with FITC EPCs after-UEA-1 for yellow fluorescence, double staining positive cells to differentiate the EPCs, show that the cultured cells of EPCs. observation can be found that "pebbles" cells in the sample, sample spiral growth. By laser confocal microscopy cells visible after dealing with the DiI-ac-LDL EPCs in red fluorescence positive, combined with FITC EPCs after-UEA-1 for yellow fluorescence, double staining positive cells to differentiate the EPCs, show that the cultured cells of EPCs. After ligation of the left hind leg femoral artery and its main branches by laser doppler blood flow ratio, regardless of the diabetes, EPCs at a high sugar or the environment, both ROS products are increased. The MDA level in diabetes group higher than healthy rats (P<0.05), and EPCs operation surgery and transplantation in rats had no effect on MDA level(P>0.05); Oxidation of diabetic rats and normal rats compared with higher levels of stress, and leads to ischemia and hind legs suppression limited recovery of blood flow, and can be reversed by oxidation inhibitor. Diabetes ROS increased under the condition of body, T-beta-catenin,to decrease relative protein(P<0.05), especially NP-beta-catenin decrease obviously(P<0.01), compared with normal group have statistical significance, and then through Licl and regulate T-beta-catenin XAV-939, NP-beta-catenin expression, can cause transplantation treatment restore blood flow change:the number of EPCs ischemic hind legs move, the number of new blood vessels and blood flow values are positively correlated and its expression quantity size, that diabetes in vivo wnt/beta-catenin signaling pathway may be associated with the EPCs treatment effect is limited. Serum Wnt5a is closely associated with diabetic ischemic lower limbs to improve blood flow, and wnt3a, wnt7a change does not conform to restore blood flow to the trend. Wnt/beta-catenin signal is low in Diabetes, the decrease of the expression of VCAM-1(P<0.05), causing EPCs adhesion ability inDM. And, in vitro, EPCs after high sugar processing,/beta-catenin signaling pathway may cause the reduce of VCAM 1(P<0.05), may lead to the ability of EPCs adhesion to the ischemic area(P<0.05). Conclusion:(1) EPCs cells at a high glucose or the diabetes have higher level of oxidative stress, which is one of the important factors leads to the inefficiency of therapeutic effect Of EPCs;(2)The WNT/beta-catenin signaling pathway in the EPCs by high oxidative stress inhibition caused decreased expression of VCAM 1, adhesion function, causing diabetes limited ischemic hindlimb to restore blood flow;And wnt5a may participate in the regulation process. |
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