Human Umbilical Cord Blood Stem Cell Transplantation For Treatment Of Diabetic Rats Limb Ischemia Research

PartⅠStudy on culture,identification and expand of CD34~+ cells from human umbilical cord bloodObjectives:The experiments aimed at the separation of high-purity CD34~+ cells from human cord blood and evaluation of its ability of amplification and surface marker.Another objective is compare the method of hespan sedimentation and centrifugation of using ficoll liquid to separate mononuclear cells in umbilical cord blood.Methods:The term umbilical cord blood of newborns was collected from the umbilical cord(UC) vein with informed consent of the healthy mother.Experimental group apply Hespan sedimentation and control group apply Ficoll density centrifugation to separate mononuclear cells.CD34~+ cells were selected and enriched from mononuclear cells by magnetically activated cell sorting(MACS),CD34~+ cells was cultured with serum-free medium contain SCF,TPO and G-CSF.Microscope were performed to observe the cells. Purity and surface marker of CD34~+ cells was determined by flow cytometry before and after culture.Results:There was no statistical difference between experiment and control group of cord blood volume and obtained mononuclear cells.Experiment group obtain significantly more CD34~+ cells than control group with(6.25±1.11)×10~9 vs(4.04±0.66)×10~9(p＜0.05). There was no statistical difference between two groups of Purity,Cell viability and multiple of amplification.The cell surface marker of CD133 and CD117 was appearanced after culture.Conclusions:The method of hespan sedimentation with magnetically activated cell sorting can obtain high-purity CD34~+ cells from human cord blood.Apply serum-free medium contain cell growth factor of SCF,TPO and G-CSF can expand CD34~+ cells in vitro. PartⅡEstablishment and Observation of Nude Mouse Diabetes Meilitus Model and lower limb ischemia ModelObjectives:To establish an animal model similar to the metabolic abnomalities of human type 2 diabetes mellitus.To establish critical lower limb ischemia model of nude mice.Methods:Forty BALB/C nude mice were randomly divided into four groups,groupⅠ: high-fatty diet that contained for 4 weeks to induce insulin resistance,then streptozotion (30mg/kg)was injected intraperitoneally and then high-fatty diet 4 weeks(n=10). groupⅡ:Ordinary diet for 4 weeks then streptozotion(30mg/kg)was injected intraperitoneally and then same diet 4 weeks(n=10).groupⅢ:High-fatty diet for 8 weeks (n=10).groupⅣ:Ordinary diet for 4 weeks then same volum citrate sodium buffer injected intraperitoneally and then same diet for 4 weeks(n=10).At 8 week groupⅠandⅣestablish the model of hind-limb ischemia.Hind limb ischemia model was established by ligation and excision of femoral artery,arteria saphena,circumflex of external iliac artery and muscular branches of femoral artery.Symptom of ischemia,temperature and Pathology of skeletal muscle was observed.Results:Four weeks after STZ injection,blood glucose and total cholestero of groupⅠsignificantly increased than groupⅡandⅣ.The evidences from optical microscope observation further confirmed pathological changes on pancreatic islets cells of diabetic models along with the diabetic symptoms(polydipsia,polyphagia,diuresis).Symptom of ischemia of groupⅠwas critical than other groups.Compare with groupⅣthe blood flow recovery more slowly in groupⅠat7,14 and 28 days(p＜0.05).HE stain find significantly disorder of muscle fiber,muscular atrophy and fibrous degeneration in groupⅠ.Conclusions:Type 2 diabetes mellitus animal model is successfully reproduced by using tIle high fatty diet plus low dose STZ injection.It may prvide a perfect animal model for investigating type 2 diabetic mechanism.Diabetes mellitus hind limb ischemia model with ligation and excision of femoral artery and it's branch is close to critical limb ischemia. PartⅢFunctional Recovery of Diabetes Mellitus ischemic Through CD34~+ stem cells transplantationObjectives:Diabetes mellitus is characterized by poor circulation and impaired angiogenesis.Administration of stem cells that can accelerate blood flow restoration to ischemic limbs of diabetic mice.Human umbilical cord blood(UCB) contains high numbers of CD34~+ stem cells.This study investigate the angiogenesis and trophic mediation effect of CD34~+ stem cells of UCB which cultured with serum free medium using a surgical-induced hind limb ischemic mouse model.Methods:Unilateral femoral artery ligation/excision was performed in 30 BALB/C nude mice of type 2 diabetes.Group 1(n=10):expanded CD34+cells 6.68×10~5/0.1 ml suspension was injected into gastronemius muscle.Group 2(n=10):Mononuclear cells of UCB 6.68×10~5/0.1 ml were was injected into the same muscle.Group 3(n=10):0.1ml of PBS+0.1%BSA was administrated into the same sites.The mice were imaged to determine perfusion recovery at 3,7,14,and 28 days post injection,and sacrificed at 12 hours and 28days later to examine mRNA and protein of TGF-β1 and CD105,mRNA was examined by real-time PCR and protein were measured by Western blot,28 days to check vascular density and labeled with human-specific monoclonal antibodies(HNA).Results:First,among three groups there was no difference of recovery of perfusion that expressed as a ratio of perfusion in the ischemic versus normal limb on day 14 post injection,however,group 1 significantly improved on day 28 after injection compare with group 2(81.0±1.42,vs 60.3±3.21 p＜0.001).Secondly,in group 1 HNA+cells were found at the edge of muscle bundle in ischemic mice at 28 days.Capillary density were significantly greater in those with CD34~+ cells injection.Thirdly,after 12 hours of administration cells TGF-β1 mRNA was significantly greater in group1 vs.group 2 and 3(0.578±0.05 vs. 0.404±0.08 and 0.239±0.09 p＜0.05).Similarly,TGF-β1 protein expression was significantly greater in the group 1 vs.other controls(20.6±2.31 vs.11.6±1.83 and 7.5±0.19 p＜0.05).CD105mRNA was significantly greater in group 1 vs.group 2 and 3(0.689±0.07vs0.504±0.08 and 0.353±0.10 p＜0.05).CD105 protein expression was significantly greater in the group 1 vs.other controls(18.1±1.96 vs.9.71±1.39and 6.0±0.10 p＜0.05). Finally,28 days later there is no difference of TGF-β1 mRNA and CD105mRNA among three groups.But TGF-β1 protein expression was significantly greater in the group 1 vs. other controls(30.9±3.05 vs.15.4±2.91 and 13.9±1.32 p＜0.05).CD105 protein expression was significantly greater in the group 1 vs.other controls(40.7±2.67 vs.19.4±3.75and 16.1±1.79 p＜0.05).Conclusions:Our study shows that direct injection of expanded UCB stem cells into the infarct zone at the time of limb ischemia induces neovascularization and improves limb temperature,which indicating an increase in limb function due to blood flow and vascularization.TGF-β1 and CD105 significantly greater shows that CD34~+ stem cells have trophic mediation.