Empirical Study Of Treating Diabetes Rabbits Ischemic Disease Of Lower Extremity With Autologous Bone Marrow Stem Cell Transplantation

Background:With the development of living standard and the advent of ageing society, disease incidence of diabetes was increasing year by year, and the enormous medical expenditure on the complication diabetic foot and amputated extremity brought about heavy economic burden. The traditional therapy includes medication, operation and interventional therapy, but the therapeutic effect is not inadequate. The technique of autologous bone marrow stem cell transplantation provides new method for curing diabetic foot.Part 1 Diabetes Animal ModelsObjective: We selects ALX to set up diabetes animal model, in order to explore a kind of safe, stable, low mortality method. Method:20 healthy rabbits were divided into 2 groups by random, and were abrosia 12 hours' before dosage. Rabbits in group A were directly given ALX 5% at 150mg/kg, and rabbits in group B were given ALX at twice, 80mg/kg of ALX 5% was intravenously injected firstly, 24 hours later, 120mg/kg of ALX 5% was given again. 14 days later, fasting blood glucose >16mmol/L means that the diabetes animal models were achievement.Results:In group A, 7 rabbits deceased within 5 days and the mortality was 70%, and 2 weeks later the fasting blood glucose of 1 rabbit recovered to the normal, then 2 rabbits were achievement and the achievement ratio was 20%;In group B, 1 rabbits deceased and the mortality was 10%, and 2 weeks later the fasting blood glucose of 1 rabbit recovered to the normal, then 8 rabbits were achievement and the achievement ratio was 80%.Conclusion:1. The twice administration method was better than the once with a higher model achievement ratio and lower mortality. 2. Selection of ALX to set up diabetes animal model was a kind of safe, economic, reliable method.Part 2 Mobilization and Effect of Bone Marrow Stem Cell Transplantation to Diabetes RabbitsObjective: According to counting the peripheral blood mononuclear cells and the bone marrow mononuclear cells and the bone marrow CFU-GM of the three groups(I.e. the group of healthy rabbits, the group of diabetes rabbits and the group of the non-diabetes rabbits)followed by mobilization with identical dose G-CSF, we observed the effect of diabetes for stem cell mobilization.According to the comparison of hemoglobin, platelet, fasting blood glucose and haemorheology before and after mobilization, we observed the effect of bone marrow stem cell mobilization for diabetes.Method:Healthy rabbits were divided into 3 groups by random. Group A were healthy rabbits, Group B were diabetes rabbits, Group C were non-diabetes rabbits (the dosage of ALX was same to group B). We counted peripheral blood mononuclear cells of the 3 groups before and after G-CSF mobilization, once a day and continued 5 days, and then the next day we draw-off bone marrow 3ml, counted bone marrow mononuclear cells, cultivated CFU-GM. We tested the counts of hemoglobin, platelet, fasting blood glucose and haemorheology of the diabetes rabbits of group B before and after mobilization, compared the diversity.Results:1. Peripheral blood mononuclear cells of 3 groups were indiscrimination before and after mobilization. Mononuclear bone marrow cells of 3 groups were indiscrimination after mobilization. 2. The counts of CFU-GM of 3 groups were indiscrimination after mobilization.3. Before and after mobilization, the fasting blood glucose general average of the 10 diabetes rabbits showed downtrend, and the hemoglobin and the platelet general average showed rise tendency. The plasma viscosity and the erythrocyte aggregation were indiscrimination before and after mobilization. The whole blood viscosities were heightened after mobilization than before.Conclusion:1. The diabetes was no effect on bone marrow stem cell mobilization.2. G-CSF mobilization was no effect on hemoglobin, platelet, fasting blood glucose, but mobilization was effect on haemorheology, which remind us to think highly of it and interfere in it.Part 3 Transplantation of Autologous Bone Marrow Stem Cell for the Treatment of Rabbits Diabetic Lowe Limb IschemiaObjective: The objective was contrast the effect on neovascularization of the three methods, which were bone marrow stem cell transplantation, hypodermic injection G-CSF and the association of above both.Method:We took 40 diabetes rabbits and ligatured arteria femoralis of the right side posterior limb to found diabetes lower limb ischemia animal models. Then the 40 diabetes rabbits were divided into 4 groups by random. Group A:We mobilized by hypodermic injection G-CSF,once a day and continue 5 days. Group B:We mobilized 5 days, On the 6 day of mobilization, we draw-off bone marrow 5ml and injected bone marrow mononuclearcell in ischemia tissue. Group C: We draw-off bone marrow 10ml without mobilization and injected like group B. Group D:We only injected PBS in ischemia tissue. 28 days latter, rabbits of 4 groups were executed. Partial musculatures were stained by the method of immunohistochemistry to detect the expression of vWF and VEGF.Results:Bone marrow stem cell transplantation and hypodermic injection G-CSF can strengthen the expression of vWF and VEGF. But when association of the both, the expression of vWF and VEGF were more strengthen.Conclusion:1. The G-CSF treatment can also be beneficial to regenerate vascular structure as the autologous BM-MNCS transplantation.2. Associating bone marrow stem cell transplantation with hypodermic injection G-CSF was better than the any single one.