| Objective: To establish a simple and stability animal model of diabetic food,observe the therapeutic effects of bone marrow stem cells(BMSCs) transplanted intodiabetic rats. Evaluate the microvessel density and the VEGF expression of ischemicmuscle tissues by immunohistochemical after treament. Detect the expression level ofAng-I by RT-PCR.Methods:1.Thirty mail Wistar rats were divided randomly into two groups:experiment group(n=25) and control group(n=5).Rats in experiment group wereintraperitoneal injected of45mg/kg streptozotocion after high fat-sugar diet for onemonths while those in control group were injected citric acid-sodium citrate bufferinstead.2.The blood glucose level was measured with test strips3days after STZinjection,and diabetes was confirmed when the blood glucose level was≥16mmol/l.3.The diabetic rats were ligated the proximal end of the femoral artery at the level of theinguinal ligament to establish the ischemic hindlimb model.4.Five healthy Wistar ratswere sacrificed and bone marrow was harvested from femur and tibia. Bone marrowmononuclear cells were isolated after density gradient centrifugation.5. PrepareBMMNC-suspension to stand.6.Diabetic foot rats(n=16) were randomly divided intothree groups as follows.In the non-treatment group(groupA,n=5),rates were not injectedat all.In the treatment group(groupB,n=6),the ischemic hindlimbs were given BMMNCstreatment as previousily described.In the sham group(groupC,n=5) the ischemichindlimbs were given PBS only.7.During the experimental period,fasting plasmaglucose,body weight,water and intake,urine in each group were observed andmeasured.The rates in all groups were sacrificed and muscle tissues were harvested forhistological detection and RT-PCR28days later. The microvessel ensity and the VEGFexpression of ischemic muscle tissues were evaluated by immunohistochemical.The Ang-I expression of ischemic muscle tissues were evaluated by RT-PCR.Results:1.3days later after STZ injection,the blood glucose level of19rats in theexperiment group(n=25) was≥16mmol/l.While they had these symptoms:excessivethirst,frequent urination,extreme hunger,unusual weight loss.7days later after the ratsnot standard with re-injection of STZ,all the rats were confirmed as diabetes.5in the21diabetic rats were sacrificed,6days later after the femoral artery ligation.We used theremaining16diabetic foot model in the second part of the experiment.12rats finishedthe whole process of experiment.Ischemic hindlimbs of all rats shrunk and activitydecline gradually after the femoral artery ligation. There’s no significant gangrene inischemic hindlimb until all rats were sacrificed.2.The expresstion of vWF in ischemichindlimb by immunohistochemistry after be transplanted localy:group A4.667±0.577，groupB7.25±0.5，groupC4.3±0.819。The microvessel quantity of group B is moreintense than that of groupA and C,P<0.05.3.The expresstion of VEGF of ischemichindlimb by immunohistochemistry after transplanted BMMNC:groupA123.55±15.70;groupB194.55±16.34;groupC120.17±14.87. The expresstion of group B ishigher than that in group A and C, P<0.05.4.mRNA expression of angiopoietion-I aftertransplanted BMMNC:groupA0.482±0.017,groupB1.173±0.028,groupC0.474±0.016.GroupB is significantly higher than that of group A and group C,P<0.05。Conclusions:1. Insulin resistance could be induced by high fat-sugar diet for4weeks.Part of the pancreatic islet βcells could be destroied by low-dose STZ(45mg/kg,intraperitoneal injection).Combining these two operations,animal models which close tohuman Type II diabetes can be induced. Diabetic rats after femoral artery ligation canbe a good simulation of the pathogenesis of diabetic foot patients with ischemicvascular occlusion.But it cannot completely reflect that diabetic foot is a systemicdisease characteristiced severe impairment.2. BMMNC could be harvested by collectingbone marrow blood and then centrifugal. After be injected in ischemic muscle tissue,BMMNC can stabilize homing to damaged tissue and then involve in angiogenesis insignificant improvement in limb blood.3. The vWF expresstion of ischemic hindlimb inexperimental group is more than the control group after BMMNC transplantion. Thedifference was statistically significant.4. The VEGF expresstion of ischemic hindlimb inexperimental group is more than the control group after BMMNC transplantion. Thedifference was statistically significant5. Ang-I mRNA extraction quality ofexperimental group was significantly higher than that of control group. The differencewas statistically significant.6. The mechanism of angiogenesis induced by BMMNCT in diabetic rates is speculated to the following:therapeutic angiogenesis was related toendothelial differentiation from the bone marrow cells and to the production ofangiogenic cytokiness,including VEGF,Ang-I,etc. They promote angiogenesis throughmultiple channels and accelerate vascular construct. |
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