| bjective:Diabetes has become the third largest serious chronic diseases threated to human health, after the cancer and cardiovascular disease. Vascular disease is one of the major complications in diabetes, as well as the main cause for disability and death. The current studies have suggested that collateral formation impaired is one of the most important reasons of diabetic vascular disease. Many pathogenesis have proposed on diabetic vascular disease, including four the mechanism—activation of polyol pathway,PKC pathway,and amino sugar pathway and formation of AGEs, for its own shortcomings, all of them can not fully explain diabetic vascular complications. Recent studies have shown that oxidative stress involved in the occurrence and development of cardiovascular disease, and Banting Award winner Brownlee.M has proposed the theory of "common mechanism of diabetic vascular complications", which points out that an excessive peroxidase in mitochondrial electron transport chain is common mechanism of vascular injury caused by hyperglycemia. the theory can reasonably explain the internal relations of oxidative stress and diabetic vascular injury in diabetic complications, as has been considered a breakthrough in the field and has caused wide public concern. Vascular disease caused by enhancing oxidative stress and vascular disease in diabetic patients have the same pathological changes and the clinical consequences, all relating to the EPC proliferation impaired and formation of new blood vessels impaired. VEGF is one of the most important angiogenesis factor, some studies once suggested that angiogenesis dysfunction is caused by VEGF reduced, but in the diabetic ischemic hindlimb model, many studies found VEGF secretion did not decrease. Many studies also found that treatment of diabetic ischemic disease with VEGF are less effective. It is suggested the rapid revascularization blocked after limb ischemia in diabetic patients has nothing to do with the VEGF. Currently, the level of oxidative stress in EPCs and the expression of VEGFR in EPCs affected by the high glucose have no studies at home and abroad, So this experiment were designed. The rat bone marrow mononuclear cells isolated by using rat lymphocyte separation medium fluid, and the cell isolated and cultured were identified applaying with DiI-acLDL and FITC-UEA-1 double staining afrer 3 days. dual fluorescence-positive cells were considered to be endothelial progenitor cells (EPCs). EPCs identified were treated with various concentrations of high glucose (terminal glucose concentration in medium 15 mmol/L,30 mmol/L,60 mmol/L respectively), to detect the proliferation in EPCs and the markers of oxidative stress in EPCs at 24h,48h,96h respectively to assess the level of oxidative stress and the proliferation in EPCs affected by high glucose. Finally, the expression of VEGFR in EPCs affected by high glucose were observed, and through comparing with antioxidant treatment, Preliminary discussing high glucose induced oxidative stress on expression of VEGFR in EPCs, to provide theoretical support for diabetic ischemic limb revascularization blocked.Methods:12 SD rats were harrested, immersed in 75% alcohol for 15 minites. Place rats on clean workbench, isolate the femur and tibia under sterile conditions, cut off the ends of long bones, expose bone marrow cavity, extract PBS containing 1% heparin with 5ml Syringe to wash the marrow cavity, use rat lymphocyte separation medium fluid to isolate the rat bone marrow mononuclear cells. the cell were identified applaying with DiI-acLDL and FITC-UEA-1 double staining afrer 3 days. dual fluorescence-positive cells were considered to be endothelial progenitor cells (EPCs). Subcultured and divided groups after 5 days, according to the various glucose concentrations,the cell were divided into two groups:the control group (terminal glucose concentration in medium was 5.5 mmol/L) and the high glucose intervention group. The high glucose intervention group were divided into three subgroups:high glucose group 1 (terminal glucose concentration in medium was 15 mmol/L glucose); high glucose group 2 (terminal glucose concentration in medium was was 30 mmol/L); high glucose group 3 (terminal glucose concentration in medium was was 60 mmol/L). The proliferation in EPCs were detected by MTT method and the markers of oxidative stress as anti-02-,MDA,GSH in EPCs were detected by the colorimetric method at 24h,48h,96h respectively. Finally, the expression of VEGFR in EPCs were detected by immunohistochemical method with and without the intervention of antioxidant therapy.Results:1. Cell morphology of EPCs was observed with light microscope, mononuclear cells just isolated were round, a little. Cultural mononuclear cells started to appear adherent growth after 2 days and started to appear colony formation after 3 days, and spindle cells and cell clusters can be seen clearly,and its structure was similar to blood island, and spindle cells were both wired -like structure and single cell. The line-like arrangement of spindle cells, and with time increasing, the cells become bigger, appearing a typical cobblestone-like changes after 7 days.2. The cultural cell appeared typical DiI-acLDL and FITC-UEA-1 double positive staining after 3 days.3. The high glucose intervention group compared with the control group at 24h point, the proliferation in EPCs were significantly enhanced in high glucose groups 3 (P<0.05); the high glucose intervention group compared with the control group at 48h point, the proliferation in EPCs were significantly reduced in high glucose group 2 and group 3 (P<0.05); the high glucose intervention group compared with the control group at 96h point, the proliferation in EPCs was significantly reduced further in high glucose group 2 and group 3 (P <0.05), and control group and high glucose group 1 have no significant difference. Different time points in each experimental group compared with the 24h point, the proliferation in EPCs was significantly enhanced (P<0.05) at the 48h and 96h point in high glucose group 1; the proliferation in EPCs has no significant difference in high glucose group 2; the proliferation in EPCs were significantly decreased at the 96h point in high glucose group 3 (P<0.05).4. The high glucose intervention group compared with the control group at 24h point, the concentration of anti-O2-in EPCs significantly increased in high glucose group 1 (P<0.05); the high glucose intervention group compared with the control group at 48h point, the concentration of anti-O2- in EPCs significantly reduced in high glucose intervention group(P<0.05); the high glucose intervention group compared with the control group at 96h point, the concentration of anti-O2- in EPCs was significantly reduced in high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the concentration of anti-O2- in EPCs significantly increased at the 48h point in the control group (P<0.05); the concentration of anti-O2- in EPCs had no significant difference in high glucose group 1; the concentration of anti-O2- in EPCs reduced in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).5. the high glucose intervention group compared with the control group at 24h point, the concentrations of MDA in EPCs had no significant difference in high glucose intervention group; the high glucose intervention group compared with the control group at 48h point, the concentrations of MDA in EPCs increased significantly in high glucose group 2 and group 3 (P<0.05); the high glucose intervention group compared with the control group at 96h point, the concentrations of MDA in EPCs significantly increased in high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the concentrations of MDA in EPCs had no significant difference in control group at the 48h and 96h point; the concentrations of MDA in EPCs increased significantly in high glucose group 1 at the 96h point (P<0.05); the concentrations of MDA in EPCs increased significantly in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).6. the high glucose intervention group compared with the control group at 24h point, the concentration of GSH in EPCs have had no significant difference in high glucose intervention group; the concentration of GSH in EPCs were significantly decreased in the high glucose group 2 and group 3 at 48h and 96h point (P<0.05); the concentration of GSH in EPCs have had no significant difference in high glucose group 1. Different time points in each experimental group compared with the 24h point, the concentration of GSH in EPCs significantly increased in the control group at the 48h and 96h point (P<0.05); the concentration of GSH in EPCs had no significant difference in high glucose group 1; the concentration of GSH in EPCs significantly reduced in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).7. (1). High glucose intervention group compared with the control group at 24h and 48h point respectively, the expression of VEGFR in EPCs significantly reduced (P<0.05) in the high glucose group 2 and group 3; high glucose intervention group compared with the control group at 96h point, the expression of VEGFR in EPCs significantly decreased in the high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the expression of VEGFR in EPCs had no significant difference in the control group; the expression of VEGFR in EPCs significantly reduced in high glucose group 1 and group 3 at the 96h point (P<0.05); the expression of VEGFR in EPCs significantly decreased in high glucose group 2 at the 48h and 96h point (P<0.05). (2). Under antioxidants therapy, the expression of VEGFR in EPCs had no significant difference in each experimental group with the time and concentration increasing. (3). Antioxidants therapy compared with non-antioxidants therapy, the expression of VEGFR in EPCs significantly increased in high glucose intervention group, in a certain extent, the serious defects of expression of VEGFR in EPCs saved by antioxidants therapy in the intervention group.Conclusion:1. High glucose significantly inhibite the proliferation in EPCs, and with the glucose concentrations and time increasing,the role of inhibition become stronger and stronger.2. Oxidative stress induced by high glucose in EPCs significantly increase, and anti-oxidation significantly decreased, with the concentration and time increasing, the role of oxidative stress become stronger and stronger.3. High glucose significantly inhibite the expression of VEGFR in EPCs, But under antioxidants therapy, expression of VEGFR in EPCs in high glucose significantly increase, in a certain extent, the serious defects of expression of VEGFR in EPCs saved by antioxidants therapy in the intervention group. Suggesting that increasing own oxidative stress in EPCs is one of the important reasons which inhibited the expression of VEGFR in EPCs.
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