Treatment Of Pulmonary Hypertension Induced By Monocrotaline In Rats By Advanced Endothelial Progenitor Cells Transplantation

ObjectiveTo study the effect of late endothelial progenitor cells(EPCs)transplantation on pulmonary hemodynamics in rats with pulmonary hypertension(PAH)induced by monocrotaline and the related mechanism of it.Method1.Cultivating,identification and labeling of the Late EPCs : After the rats were sacrificed,their femurs and tibia were rapidly separated and the bone marrow cavity was rinsed under sterile conditions.EPCs were isolated and cultured by density gradient centrifugation and poor adherence method.Through the Inverted microscope,the growth and morphological changes were observed..After incubation for 21 days,Dil-labeled acetylated low density lipoprotein(Dil-ac-LDL)and FITC-labeled venomin 1(FITC-UEA-1)were stained with double fluorescence and the cell surface antigen was identified by immunophenotype CD133,v WF,FIK1 expression.After identification of the cells,late doses of EPCs were labeled with the dye PKH26 at a concentration of 4 x 10-6 M,which was ready for transplantation.2.Experimental grouping and animal model preparation: Fifty male Wistar rats were randomly divided into model group,blank group,Shenfu Yixin granule group,endothelial progenitor cell group and combined group(Shenfu Yixin granule + endothelial progenitor cells).The rats that in the model group,Shenfu Yixin granulegroup,endothelial progenitor cell group and combined group were treated withsubcutaneous injection of 1% monocrotaline solution into the back of the neck by 50 mg / Kg,to make the pulmonary hypertension model.While rats in the blank group did not interfere.After 28 days,the rats were treated with Shenfu Yixin granules(according to their clinical dosage of 205 g daily,60 Kg body weight according to the conversion coefficient of 6.25,and finally the daily dosage of rats was calculated.21.35g/kg),blank group,model group and endothelial progenitor cell group were given equal volume of normal saline every day for 28 days.3.EPCs transplantation: After the model was established,the endothelial progenitor cells and the combined group were injected with 500 ?l of PKH26-labeled EPCs via the jugular vein.The cell density was 2 × 106 cells / ml,and the other three groups were injected with the same amount of EBM2.After transplantation,the five groups continued to give the corresponding drug or saline for 4 weeks.4.Measuring the mean pulmonary artery pressure,cardiac output and right heart hypertrophy index: 28 days later,the modified right ventricular systolic pressure RVSP right ventricular diastolic pressure RVDP and mean pulmonary artery pressure m PAP were measured using the modified PE-50 catheter.And then indirectly calculated the rat pulmonary vascular resistance PVR After the pulmonary artery pressure was measured,the cardiac output of the rats was measured by thermal dilution.Calculating the right heart hypertrophy index RV /left ventricular + ventricular septal(LV+S)5.Tracked in lung tissue of the EPCs: Take the lungs of the right lung door of the rats to take specimens,made frozen tissue sections.The expression of PKH26-labeled endothelial progenitor cells in lung tissue was observed under fluorescence microscope.6.Detection of paracrine of EPCs by ELISA: After 4 weeks of drug intervention,rats were sacrificed and serum was collected.The levels of stromal cell-derived factor-1(SDF-1),hypoxia-inducible factor(HIF-1)and vascular endothelial growth factor(VEGF)were measured by ELISA.7.Detection of EPCs related growth factors and apoptosis-related genes by q PCR method: The expression of EPCs-related growth factor and apoptosis-related genes were detected by q PCR,including stromal cell-derived factor-1(SDF-1),hypoxia-inducible factor-1(HIF-1),vascular endothelial growth factor(VEGF)and apoptosis-related factor Bcl-2,Fasl.Result1.After 5 days of culture,the cells began to appear linear or blood-like structure.After 14 days,the linear and blood-like structure began to disappear gradually,with the proliferation of cells was paving-like structure.Until 21 days,the cells were mainly paving stones,and gradually covered the board.Inverted fluorescence microscopy showed that EPCs ingested Dil-ac-LDL as red fluorescence,and FITC-UEA-1 was green fluorescence,and double-stained positive cells were orange-yellow.CD133,v WF,FIK1,both showed positive markers.After staining with PKH26,the red fluorescence was observed under the microscope.2.The results showed that m PAP,RVSP,RVDP,RV /(LV + S)and PVR were significantly higher in the model group than in the blank group(P <0.05),while the CO and CI were lower(P <0.05).The levels of RVSP,RVDP,m PAP,RV /(LV + S)and PVR in the EPCs group,Ginseng Yixin granule group and the combined group were lower than those in the model group(P<0.05),while the CO and CI increased(P<0.05).Compared with EPCs group(P> 0.05),there was no statistical significance.3.Detection of paracrine of EPCs by ELISA :Compared with the blank group,the levels of SDF-1,HIF-1 and VEGF in plasma were significantly higher(P <0.05).Compared with the model group,the levels of SDF-1,HIF-1 and VEGF in EPCs group,Ginseng Yixin granule group and combined group were significantly higher than those in model group(P <0.05).The levels of SDF-1,HIF-1 and VEGF in plasma of pulmonary hypertension rats were significantly increased after endothelial cell transplantation The combined group was significantly higher than the Ginseng Yixin granule group and EPCs group.4.Detection of EPCs related growth factors and apoptosis-related genes by q PCR method.Compared with the blank group,the levels of VEGF,SDF-1 and HIF-1 in the other four groups were significantly higher than those in the blank group(P <0.05)Compared with EPCs group and the Ginseng Yixin granule group,the levels of VEGF,SDF-1 and HIF-1 in lung tissue in combined group were significantly increased(P <0.05).Compared with the blank group,the Fasl in the lung tissue of the model group was significantly higher.Compared with the model group,the Fasl content of the Ginseng Yixin granule group,EPCs group and combined group was significantly decreased,and the combined group was significantly lower than the Ginseng Yixin granule group and EPCs group(P<0.05).Compared with the blank group,the Bcl-2in the lung tissue of the model group was significantly decreased.Compared with the model group,the content of Bcl-2in the Ginseng Yixin granule group,EPCs group and combined group was significantly higher and the combined group was significantly higher than the Ginseng Yixin granule group and EPCs group.Conclusion 1.EPCs transplantation can improve pulmonary hemodynamics in PAH rats,reduce right heart hypertrophy index,and improve heart failure.2.Shenfu Yixin granule can improve pulmonary hemodynamics in PAH rats,reduce right heart hypertrophy index,and improve heart failure.3.EPCs may improve pulmonary hypertension by secreting VEGF,SDF-1,HIF-1 and other related growth factors and regulating Fasl,Bcl-2cell apoptosis genes.