| Abstract Objectives The complications of diabetes become a great threat to human health and is a worldwide public health problem.Diabetic vascular complications with a high incidence, can reslut in lower limb ischemia, foot ulcers, even amputation.It is an important factor in disability to the patients.This has brought great pain and burdens to patients and their families.The pathogenesis of diabetic lower limb ischemia is complicated and is associated with atherosclerosis with collateral circulation damage, which accounts for impaired collateral vessels capable of forming. In recent years, a large number of studies' have shown that EPC (endothelial progenitor cells) play a prominent role in the formation of blood vessels.The relative small number of circulating EPC was found under normal circumstances, whereas it increases tremendously in the trauma or ischemia. However, EPC proliferation in patients with diabetes is much lower than that in non-diabetic patients.Its adhethsion, proliferation to activation of endothelial,and the ability to form small blood vessels are reduced. The response of ischemic tissue's to exogenous growth factor is also reduced. Endothelial progenitor cells exist in umbilical cord blood and the number of EPC is much higher than that in peripheral blood. Collection EPC in umbilical cord blood is non-invasive, easy to find the consistency of unrelated donor HLA. Using Endothelial progenitor cell for treatment of diabetic vascular disease is a field worthy of study, but research in this respect is lack both in China and other countries.Therefore,we used streptozotocin (STZ) to establish diabetes model and ligated the femoral artery to form double-hind limb ischemia. The application-ray irradiation is used to reduce immune rejection. Maternal human cord blood was isolated and cord blood mononuclear cells cultured for 7 days to gain endothelial progenitor cell.The endothelial progenitor cells were transplanted into rats by tail vein injection or local injection. By observing the rat hind limb ischemia and ulceration, fluorescent tracer was used to trace endothelial progenitor cells with GFP.Ⅷfactor and VEGF expression in the study were measued to explore the efficiency of the umbilical cord blood endothelial progenitor cells to treatment of diabetic lower limb ischemia as well. Methods 1.Isolation, culture,identification endothelial progenitor cells:first taking maternity full-term umbilical cord blood 50ml,use density gradient centrifugation to gain mononuclear cells, purify endothelial progenitor cells to culture 7 days by its characteristics of adherent growth.Then the cultured cells were digested, and identified by flow cytometry with CD133,CD34, KDR antibody and counted the number of living cells.Viable cells number were determined.2.establish diabetic rat model and divide groups:The 20 healthy male Wistar rats(weighing about 200-250g)were fed for a week, later 15 were randomly selected as diabetes groups,5 for the normal blood glucose in rats given injection of citric acid buffer. Diabetes groups were given single intraperitoneal injection of streptozotocin(STZ) 55mg/kg,all the rats were divided into four groups:①Intravenous EPC transplantation in the treatment of diabetic group (Diabetes+ligation+intravenous injection, DLV):Diabetic rats after the ligation of artery of hind are given ray irradiation to establish ischemia model.Then EPC is injected by tail vein. The efficiency of intravenous injection of EPC to treat lower limb ischemia was observed.②Local EPC transplantation in the treatment of diabetic(Diabetes+ ligation+intramuscular injection, DLM):diabetic rats after ligation of the posterior limbs of radiation, the right hind limb injection of EPC, the efficacy of the intramuscular injection of EPC treat lower limb ischemiawas observed. Local injection of equal volume of the left hind phosphate buffer (PBS) as a femoral artery ligation in treatment of diabetes control group.(Diabetes+ligation+control, DLC)③Diabetes control without injection and ligation(DC)④Normal rats with ligation and EPC transplantation group (Normal+ligation+intravenous injection, NLV):Normal rats after irradiation sterilization pairs of hind legs,tail vein is injected EPC.3.After injection of progenitor cells, following experiments were conducted:①fluorescent tracer progenitor cells:use the GFP (green fluorescence) transfect endothelial progenitor cells before transplantation, as the.transfected is success, then transplant into rats, after a week take gastrocnemius muscle tissue as frozen sections, to observe whether the fluorescence express and compare the fluorescence intensity and to trace whether the endothelial progenitor cells arrive the ischemia parts.②gastrocnemius HE staining and immunohistochemistry ofⅧfactor to measure the number of capillaries:after the injection of endothelial progenitor cells at 21 days, rats were killed and take gastrocnemius to sterile, fix, dehydrate, embed in paraffin,HE and SP staining.then count the capillary number of cases of muscle fiber to explore whether the collateral circulation is improved after treatment of endothelial progenitor cells in diabetic lower limb ischemia model.③RT-PCR detection of VEGF mRNA expression in the posterior limbs of the gastrocnemius:after the injection of endothelial progenitor cells at 21 days,rats were killed, take aseptic gastrocnemius, detect VEGF mRNA expression by RT-PCR to study the efficacy of endothelial progenitor cells to treat diabetic lower limb ischemia.④Statistical analysis: application of statistical software to analyze data SPSS 13.0,measurement data to mean±standard deviation((?)±s),between diabetic lower limb ischemia inject endothelial progenitor cells by tail or by local injection, diabetic not ischemia, normal ischemia inject endothelial progenitor cells and diabetic lower limb ischemia not inject use the single factor analysis of variance and SNK q test, diabetes local injection and intravenous injection of endothelial progenitor cells using the paired mean comparison, P<0.05 indicated statistical significance. Results 1. identification of umbilical cord blood progenitor cells:flow cytometry determin the cells cultured for 7 days, the highest content of cell was CD34, KDR levels was lowest.this show our cultured cells are early progenitor cells.2.changes of ischemic posterior limbs in every group: In diabetic model group,compare to the normal control group, apparent polydipsia, polyuria, Diy hair were observed after the STZ injection two days.From femoral artery ligation to 21 days, the recovery of ischemia and ulcer in the hind limbs with the injection of stem cells were faster than the non-injected. The recovery in goup by tail vein injection and the local injection have little difference. The recovery in diabetic rats and normal rats are not significant different.3.fluorescent tracer: gastrocnemius muscle with stem cells is bright.The expression of fluorescence in tail vein injection and local injection are similar and local injection are lower.There is no fluorescent in rats without injection stem cells(DC).4,gastrocnemius capillary observation by HE staining and Immunohistochemistry ofⅧfactor:the mean capillary density of each group are 4.75±0.957(DLC),18.5±8.737(DLM),18.75±4.349 (DLV),21±2.944(NLV),17±3.162(DC).the number of capillaries in ligated rat by gastrocnemius injection of EPC are more than that in DLC and the differences are statistically significant (P<0.05).The difference in tail vein injection and local injection is not significant(P> 0.05).5.VEGF mRNA expression in earch group:VEGF mRNA-2△△CT mean in earch group are:DLC(1.029±0.293),DLM(2.707±0.946),DLV (2.621±0.414),NLV(3.121±1.734),DC(3.414±0.677).Compare with the non-injected group, VEGF expression in injection of stem cells is increased. The expression in tail vein injection group and local injection group was no significantly different(P>0.05).diabetic in tail vein injection group and normal ischemia inject endothelial progenitor cells are no diffirence(P>0.05).Conclusions 1.Transplantation of umbilical cord blood endothelial progenitor cells for treatment of diabetic lower limb ischemia is effective.2.Therapeutic effects of umbilical cord blood progenitor cells for the treatment of diabetic lower limb ischemia are not significantly different between tail vein injection and local injection. |
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