Empirical Study Of Diabetes Rats Bone Marrow Endolthelia Progenitor Cells Culture In Vitro

Background:Diabetes are the metabolism syndrome that take the hyperglycemia as main performance,the hyperglycemia can promote the vasculopathy which due to organ organization ischemia.Endthelial progenitor cells(EPCs)are precursor cells of angiogenesis,can promote angiogenesis and the establishment of collateral circulation, they will provide new material for the treatment of ischemic disease. However,because of the small number of EPCs in vivo,furthermore diabetes disease influence EPCs on the function and the number,the clinical application of EPCs is restricted.This study sought to establish model of diabetic rats to explore the impact of diabetes on EPCs, investigate the better conditions of EPCs amplification in vitro.these will provide a theoretical foundation for the clinical application of EPCs.Part 1 Diabetes Rats ModelsObjective: We selects STZ to set up diabetes animal model, in order to explore a kind of safe,stable, low mortality method.Method:20 healthy rats were divided into 2 groups by random,and were abrosia 12 hours before dosage.Rats in group A were directly given STZ at 50mg/kg,and rats in group B were given STZ at twice,10mg/kg of STZ was intravenously injected firstly,24 hours later,50mg/kg of STZ was given again. 72 hours later,fasting blood glucose >16.7mmol/L means that the diabetes animal models were achievement.Results:In group A, 1 rat deceased within 72 hours,another one two weeks later dies and the mortality was 10%,2 rats have not become diabetes rats,then 16 rats were achievement and the achievement ratio was 80%;In group B,1 rat deceased within 72 hours, the 5th day dies 2 rats (one has not become diabetes rat)and 1 rats deceased after two weeks,the mortality was 20%, and 72 hours later the fasting blood glucose of 1 rat has not become diabetes rats,then 16 rats were achievement and the achievement ratio was 80%.Conclusion:1.compared Single dose administration with the two,model achievement ratio and mortality have not Significance differen- ce.2. Selection of STZ to set up diabetes animal model was a kind of security,the long-term stability of high,animal mortality rate low method. Part 2 Culture and identification of Healthy Rats Endolthelia Progenitor CellsObjective:when rat bone marrow EPCs culture in vivo,we add to Recombinant human granulocyte colony stimulating factor ( rhG-CSF ) and Recombinant human erythropoietin(rhEPO)with different concentration.According to monitoring the quantity and function of EPCs,we discuss whether rhG-CSF and rhEPO play a positive role on EPC culture in vivo,thereby seeking the better conditions which promote EPC growth.Method:10 health Wistar rats,Under ether inhalation anesthesia killed off neck,bone marrow was collected from the femur and tibia and placed in phosphate buffered saline.MNCs were separated using Lymphocyte separating medium (1.077 g/mL) density gradient centrifugation.we use NH4CL to digest residual erythrocytes(red blood cell ),culture in DMEM medium supplemented with VEGF20ng/ml,bFGF10ng/ml and 20% fetal bovine serum,Nonadherent cells were removed with medium change after 72 hours,Culture medium was replaced Once every three days.in the cell culture 7th day,with 0.25% trypsin cell chip climb,we identify cell phenotype(CD34, CD133, Flk-1) by Immunohistochemistry;at the same time,we inoculated the same number of cells into 96-well plates,randomly divided into control group,rhG-CSF group and rhEPO group (CK group, G group and E group),the experimental group was divided into (G1 group, G2 group, G3 Group, G4) and (E1 group, E2 group, E3 Group, E4 group),respectively add rhG-CSF and rhEPO different concentration (8,40,200,1000ug/L), (20,100,500,2500 IU/L), continue to cultivate 24h, then detected the quantity and cell adhesion function.Results:1.Bone marrow mononuclear cells which were obtained by density gradient centrifugation and NH4CL digestion Contains EPCs,which have endothelial differentiation potential,can achieve vitro expansion of EPCs.2.Both rhG-CSF and rhEPO Play a positive role in cultivating EPCs,rhG-CSF of 1000ug/L and rhEPO of 500IU/L groups have the best promoting function on quantities of living cells and adherent cells.Conclusion:1.Endothelial progenitor cells can be cultured,purification and amplification in appropriate conditions in vitro,suggesting that may meet the clinical treatment.By means of amplification in vitro,EPCs may achieve the required quantity in clinical treatment.2.rhG-CSF,rhEPO can be by way of dose-dependent manner to promote the number and function of EPCs in vitro,which has potential clinical value. Part 3 Culture and identification of Diabetes Rats Endolthelia Progenitor CellsObjective: when rat bone marrow EPCs culture in vivo,we add to rhG-CSF and rhEPO with different concentration.Accor- ding to monitoring the quantity and function of EPCs chang, we discuss whether rhG-CSF and rhEPO play a positive role on EPC culture in vivtro,thereby seeking the better conditions which promote EPC growth.Method: 30 healthy rats were divided into 3 groups.Group A were healthy rats,Group B were diabetes rats, Group C were diabetic rats group of healthy blood glucose(Insulin therapy), Cell culture methods reference part 2,in the cell culture 7th day, digest adherent cells by 0.25% trypsin,we identify cell phenotype(CD34,CD133,Flk-1)by Immunohistochemistry;at the same time,three groups of cells in each group were randomly divided into non-drug group (add the same amount of PBS solution) and the drug group (add 500IU/L of rhEPO),we inoculated the same number of cells into 96-well plates,then detected the quantity and cell adhesion function.Results:1. As far as quantity and function of EPCs is concerned,there were no statistical significance between diabetic rats group of healthy blood glucose and healthy rats group.However,they compared with diabetic rats(hyperglycemia),the numbers and function of EPCs from diabetic rats group were remarkably decreased(p<0.05).2. RhEPO improves the number and function of EPCs from both healthy rats and diabetes rats.Conclusion:1.Diabetes high blood sugar can influence the number and function of EPCs in vitro, suggesting that blood glucose control is crucial if we want to obtain appropriate EPCs from diabetic individuals.2.RhEPO Plays a positive role in culture of EPCs from both healthy rats and diabetes rats.this is application Value about rhEPO.