| Objective: The pathological changes of diabetes peripheral vascular lesions is atherosclerosis,aside the patients' high prevalence rate,the incidence of early age,the rapid disease progression,multiple organs are often involved at the same time.Endothelial cell damage is one of the initiating factors of diabetes vascular lesions.Endothelial cells can excrete a variety of cell factor,and regulate vascular tone and the permeability of vessel walls through complex mechanisms,and meanwhile produce extracellular matrix proteins,which influence the formation and remodeling of the vascular.Arterial interventional therapy has obvious efficacy in the treatment of diabetics with lower extremity ischemic disease,however,the intimal injury which is induced by the interventional treatment is one of the primary reasions of postoperative restenosis,and how to prevent the restenosis after interventional treatment is urgent.The aim of this study was to: 1 By the model of balloon dilatation of rabbit hind limbs arterial intimal injury,perform transplantation of umbilical cord blood stem cell after damaging the endarterium local.2 By monitoring,before and after the transplantation,the changes of vascular endothelial growth factor(VEGF),endothelin(ET-1)and nitric oxide(NO)level,to observe the affections of the transplantation of human umbilical cord blood stem cells on endothelial function.3 To observe the effections of the transplantation of human umbilical cord blood stem cells on repairing the arterial intima,and discuss the effectiveness of the transplantation in preventing the restenosis after interventional treatment.Methods: Experiment objects are 18 white rabbits from New Zealand,provided by The Fourth Affiliated Hospital of Hebei Medical University,Animal Experimental Center,clean grade,male,body weight 2.1 ± 0.5Kg.Randomly divided into three groups,each group of 6.Normal control group(group A),only have femoral artery separation on the left side;balloon injury group(group B),only have on the left side the femoral balloon expansion and pull,causing arterial intimal injury;group of transplantation of umbilical cord blood stem cells after balloon injury(group C),have transplantation of human umbilical cord blood stem cells in the injured left femoral artery,at the same time,group A and group B injected the same amount of PBS buffer injection as control,suture the wound after the operation,muscle injection of penicillin sodium(160 million U per time,2 times per day)for 3 days consecutively,use heparin sodium injection 200U/Kg.d for 3 days consecutively.Collect peripheral blood from each group before the operation,1 week after the operation,and 4 weeks after,determining VEGF and ET-1 level using ELISA method,determining the plasma NO using nitrate reductase method;four weeks after the operation,take the femoral artery on the left side of the animals before all of them to be sacrificed,and HE staining is performed to observe the endometrial repair.1 Umbilical cord blood stem cell collection,separation,preparationUmbilical cord blood was derived from healthy maternals.Collecting fresh umbilical cord blood(6 hours out of),diluted with PBS liquid of the same volumn and put into the centrifuge tube(50ml),adding human lymphocyte separation liquid at the ratio of 1:1,kept under 4?,8cm centrifugal radius,2000r/min.After centrifugation for 20 minutes,umbilical cord blood was divided into 5 layers,mononuclear cells in the middle layer of tunica albuginea.Using pipette to transfer the middle layer of tunica albuginea,a suspension containing mononuclear cells,carefully,into another centrifuge tube,kept under 4?,8cm centrifugal radius,1000r/min,centrifuged for 15 minutes,washed 3 times using PBS liquid.Suspended with the low sugar DMEM nutrient solution,the cell concentration counted as 5 * 105/ml.So got HUCBSC suspension.2 Preparat balloon injury modelInjected 3% sodium 2ml/kg from rabbit ear margin vein to anaesthetize,and during the surgery injected heparin 200u/kg as anticoagulant,from the left inguinal ligament to the knee joint made the longitudinal incision,separated the left femoral artery,clipped proximal and distal vascular,punctured the proximal end of femoral artery.Group A: only performed the left hind limb femoral artery dissection;Group B and group C: after puncturing,sent in 3mm-40 mm balloon slowly into to the femoral artery,added up to 12 atmospheric pressure to fill the balloon,maintained for 3 minutes,waited for 1 minute and repeated the operation,totally 3 times,and stretched the balloon,to make a left hind leg of the rabbit femoral artery balloon injury model.3 transplanting umbilical cord blood stem cell after balloon injuryFor group B and C of rabbits,after the left hind limb femoral artery balloon injury model was successfully prepared,restored blood flow for 3 minutes,clipped distal vascular of the seprated femoral artery in all three groups.For group A and B,injected 0.5ml PBS at proximal vascular of femoral artery that was clipped,for group C,injected 0.5ml human umbilical cord blood stem cells mixed suspension at proximal vascular of femoral artery that was clipped.After injection,clipped all three groups the proximal vascular of the isolated femoral artery with vascular clamp for 30 minutes,allowing the transplant stem cells fully home,sutured the wounds,restored blood flow of femoral arteries.Injected heparin(200 u/kg.d)as anticoagulant for 3 days.4 Before and after transplantation,measuring vascular endothelial growth factor(VEGF),endothelin-1(ET-1),nitric oxide(NO)levelCollected peripheral blood 6ml from group A,B,and C three times,once before the operation,once 1 week after,and a third time 4 weeks after,sodium citrate as anticoagulation,separated the plasma for use.Determining VEGF and ET-1 using ELISA method,the plasma nitric oxide(NO)using nitrate reductase method.5 Observing endometrial repairment of the damaged blood vesselsFour weeks after transplantation,made lens tissue slice of the rabbits' injured vessel using conventional method,performing hematoxylin-eosin staining,taking pictures and observing the endometrial repairation of the damaged blood vessels.With the SPSS14.0 application for statistical data analysis,data using (?)±s format,using variance analysis(F test)test between groups and in one group,that P < 0.05 was statistically significant.Results: 1 Observing of experimental animals Three groups,A,B,C,of animal gradually healed and no erosion,ulcer,normal active,after the wound,when taking the femoral artery before the animals were killed,no local gangrene.None animal quits during the experiment.2 Determining the results of VEGF level The preoperative VEGF level of three groups,A?B and C,had no statistical significance(P>0.05);one week after the surgery,the level of VEFG of group C increased,higher than that of group A and B,the difference was statistically significant(P<0.05),the level of VEFG of group B was higher than that of group A,the difference was statistically significant(P<0.05);four weeks after the surgery,the level of VEFG of group C still higher than that of group A and B,the difference was statistically significant(P<0.05).The level of VEFG of group B was higher than that of group A,the difference was statistically significant(P<0.05).The level of VEFG in group A the difference that before the surgery and one week after the surgery and that four week after the surgery was no statistically significant(P>0.05);The level of VEFG in group B The VEFG level four weeks after the surgery was higher than that before the surgery or one week after the surgery,the VEFG level one week after the surgery was higher than that before the surgery,the difference was statistically significant(P<0.05);The level of VEFG in group C The VEFG level one week after the surgery was higher than that before the surgery or four weeks after the surgery,the difference was statistically significant(P<0.05),the VEFG level four weeks after the surgery was higher than that before the surgery,the difference was statistically significant(P<0.05).3 The change of the levels of ET-1 and plasma NO Before the surgery,the difference of ET-1 level in three groups was no statistically significant(P>0.05);one week after the surgery,the ET-1 level in group C was lower than that of group A and B,the difference was statistically significant(P<0.05),the ET-1 level in group B is lower than that of group A,the difference was statistically significant(P<0.05);Four weeks after the surgery,the ET-1 level in group C is lower than of group A and group B,the difference was statistically significant(P<0.05),the ET-1 level in group B is lower than that of group A,the difference was statistically significant(P<0.05).The ET-1 level of group A the difference that before the surgery and one week after the surgery and that four week after the surgery was no statistically significant(P>0.05);The ET-1 level of group B The difference was statistically significant(P<0.05)in each pair of the three time,before the surgery,one week after the surgery and four weeks after the surgery;The ET-1 level of group C There was no difference between the ET-1 level before the surgery and that four weeks after the surgery,the ET-1 level four weeks after the surgery was lower than that one week after the surgery,was statistically significant(P<0.05).Before the surgery,the difference of NO level in the three group A,B and C was no statistically significant(P>0.05);one week after the surgery,the NO level of group C was higher than that of group A and B,was statistically significant(P<0.05),the difference of NO level between group A and B was not obvious;four weeks after the surgery,the NO level of group C was higher than that of group A and B,was statistically significant(P<0.05),the difference of the NO level between group A and B was not obvious.In group C,the NO level four weeks after the surgery was higher than that before the surgery or that one week after the surgery,was statistically significant(P<0.05).The NO level in group A the difference that before the surgery and one week after the surgery and that four week after the surgery was no statistically significant(P>0.05);The NO level in group B The NO level before the surgery was higher than that one week after the surgery or that four weeks after the surgery,was statistically significant(P<0.05),the NO level one week after the surgery was higher than that four weeks after the surgery,was statistically significant(P<0.05);The NO level in group C The NO level four weeks after the surgery was higher than that before the surgery or one week after the surgery,was statistically significant(P<0.05),the NO level before the surgery was higher than that one week after the surgery,was statistically significant(P<0.05).4 Observing femoral artery intima restoration.Four weeks after the surgery,in Group C,there was a mild hyperplasia and interrupt in femoral artery,slight fractures of elastic membrane;in Group B,there was continues interrupt in the animals' femoral artery intima,intima hyperplasia was obvious,obvious ruptures of elastic membrane;in Group A,femoral artery intima was complete,elastic membrane was continuous,no fault.Conclusions:1 Rabbit left femoral artery injury model was built through the balloon expansion blocking the bloodstream in this experiment.2 The transplantation of human umbilical cord blood stem cell,may promote rabbit model serum VEGF level,induce angiogenesis and enhance the ability of endothelial cell proliferation.3 The transplantation of human umbilical cord blood stem cell is helpful in increasing rabbit model serum NO level,reducing the ET-1 level,thereby reducing the vascular contraction,promoting re-endothelialization of damaged intima,improving endothelial function and preventing vascular restenosis.4 The transplantation of human umbilical cord blood stem cell can repair the damaged rabbit vascular intima. |
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