The Effect Of Endothelial Progenitor Cell Perfusion On Prolong Cardiac Allograft Survive With Immune Tolerance

Part 1．Isolation, Culture and Identification of EndothelialProgenitor Cells from Rat Bone MarrowObjective To investigate the methods of isolating and culturing endothelial progenitorcells (EPCs) from rat bone marrow.Methods The mononuclear cells were isolated from rat bone marrow by densitygradient centrifugation and cultured for 48h. Then the wall attached cells werecultured in the selective culture medium (with the supplement of VEGF, bFGF, IFG-1and EGF) for 2w. The cells were stained with Dil-ac-LDL and FITC-UEA-1 foridentification.Results The attached cells stretched, differentiated, and exhibited the colonalmorphology after inducing culture for 3 to 5 days. The cells proliferated faster after7-10 days' incubation when cord-like structure was observed. After 2 weeks'induction, most of the cells exhibited multi-angular morphology. More than 70% cellswere stained positively with Dil-ac-LDL and FITC-UEA-1 (double positivefluorescence).Conclusion EPCs were enriched in rat bone marrow and may exhibit some of thecharacteristics endothelial cells owned after inducing culture in vitro.Part 2．Mixed Lymphocyte Culture (MLC) with EPCsObjective To investigate the immunogenity and immune regular ability of endothelialprogenitor cells (EPCs) derived from bone marrow through mixed lymphocyteculture.Methods Splenic lymphocytes from SD rat were used as activate cells and spleniclymphocytes from Wistar rat as reactive cells. The study was composed as 5 groups.GroupⅠ, the control group, was mixed culture of 1×10~5 activate cells and 1×10~5 reactive cells. GroupⅡwas mixed culture of 1×10~4 EPCs from SD rats and the sameamount of reactive cells as GroupⅠ. GroupⅢwas mixed culture of 1×10~4 EPCsfrom SD rats and the same amount of active and reactive cells as GroupⅠ. GroupⅣismixed culture of 1×10~3 EPCs and the same amount of active and reactive cells asGroupⅠ. GroupⅤis mixed culture of 1×10~4 EPC from SD rat and the same amountof active and reactive cells as GroupⅠplus phytohemagglutinin (PHA) as astimulator. Each group was cultured for 120h. The number of lymphocyte in eachgroup was calculated with liquid scintillation counter.Results The CPM (Count Per Minute) value were 11970．2±2202．2 in GroupⅠ,1279．2±159．8 in GroupⅡ, 1194．6±362．3 in GroupⅢ, 1784．4±568．08 in GroupⅣ,4839．2±1328．2 in GroupⅤ. The cells in GroupⅡ,Ⅲ,ⅣandⅤwere less than those inGroupⅠ(P<0．01) . The cells in GroupⅡ,ⅢandⅣwere less than those in GroupⅤ(P<0．05) .Conclusion Ex vitro, endothelial progenitor cell have hardly immunogenic and havesome extent immune inhibit ability without stimulator as PHA.Part 3．Establishment of Heterotopic Heart Transplantation Model inRatsObjective To establish the methods of allograft heart transplantation in rats.Methods Thirty pairs of Wistar rats and SD rats were composed. After anesthesia andheparinization, SD rat heart was harvested as donor. The aorta of donor heart wasanastomosed to the abdominal aorta of Wistar rat as recipient with end to side fashion,and donor pulmonary artery was anastomosed to inferior vena cava of the recipientwith the same fashion. The animals were daily inspected and the survival days werecounted.Result After short time training, the transplantations were totally successful. Theaverage heart rate was 148±24．8 beats per minute and the average survival time ofallograft was 6．7±1．1 d.Conclusion The heterotopic heart transplantation model we established wasconvincable as a model of allograft heart transplantation.Part 4．The Safety of EPC Perfusion in Heart TransplantationObjective To evaluate the safety of EPC donor heart perfusion in different celldensity.Methods Fifteen of Wistar rats and SD rats were randomizely divided into threegroups (5 pairs in each group). The animal transplantation model in Part 3 was used.In GroupⅠ, the donor hearts were perfused with 1×10~7 EPC(dyed with DAPI)from theSD rats through allograft coronary artery before implantation. In GroupⅡ, the donorheart were perfused with the 1×10~6 EPC. In GroupⅢ, the donor heart were perfused with the 1×10~5 EPC.The rate of arrhythmia and myocardial infarction were observed.The allograft hearts were extracted after 5 d for fiuoroscope to calculate thefluorescent cell density.Results In GroupⅠ, three animals were developed arrhythmia and two had heart arrest.There was no arrhythmia and heart arrest in GroupⅡandⅢ(P<0．05) .There was4．73±0．69 /mm~2 fluorescent cells in GroupⅠ, 3．58±0．62 /mm~2 in GroupⅡand1．11±0．24 /mm~2 in GroupⅢ. The fluorescent cells in GroupⅢwas the least amongthree groups (P<0．05) .Conclusion The optimal cell density for donor heart perfusion was 1×10~6．Part 5．The Survival Time after Allograft Heart Transplantation withEPC PerfusionObjective To study the effect of EPC perfusion on survival time of cardiac allograftand acute rejection in heart transplantation.Methods Twenty pairs of Wistar rats and SD rats were randomizely divided into fourgroups (5 pairs in each group). The animal transplantation model in Part 3 was used.GroupⅠwas set up as the control group. In GroupⅡ, the rats was fed with CsA aftertransplantation for 7d.In GroupⅢ, the donor heart were perfused with 1×10~6EPCsbefore implantation. In GroupⅣ, the donor heart were perfused with 1×10~6EPCsbefore implantation and the rats was fed with CsA after transplantation for 7d. Theallograft heart was extracted at the time point when allograft heart was beating slowlyand weakly. The sample was cut for pathology study.Results The average donor heart survival time was 6．16±0．75d in GroupⅠ,12．5±1．05d in GroupⅡ, 10．2±1．3d in GroupⅢand 19．3±1．9d in GroupⅣ. Thesurvival time in GroupⅡ,ⅢandⅣwere prolonged more significantly than GroupⅠ(P<0．01) . The survival time in GroupⅣwas the longest among four groups (P<0．01) .Conclusion EPC perfusion with CsA administration could prolong the survival timeof the allograft heart.Part 6．Inhibition of Acute Rejection of Allograft HeartTransplantation in Rats by Donor EPC PerfusionObjective To explore the effect of donor EPC perfusion on inhibition of acuterejection in heterotopic allograft heart transplantation in rat.Methods Ten Wistar rats (5 pairs) were composed to GroupⅠ(Isograft Group). Thentwenty pairs of Wistar rats and SD rats were randomizely divided into four groups (5pairs in each group, Allograft groups). The animal transplantation model in Part 3 was used. In GroupⅡ, there was no EPCs perfusion and CsA feeding. In GroupⅢ, ratswere fed with the CsA after transplant for 7days.In Group IV, donor hearts wereperfused with 1×10~6 EPC before implantation. In GroupⅤ, the donor hearts wereperfused with 1×10~6 EPCs and the rat were fed with CsA for 7 days aftertransplantation. On the seventh day after transplantation, the rats were sacrificed andthe spleen and peripheral lymphoid nodes were taken out for CD3, CD4 and CD8 Tcell counting and CD4/CD8 ratio. Meanwhile, allograft hearts were extracted forpathological study.Results In spleen as well as peripheral lymphoid nodes, the percentage of CD3 andCD4 cells were significantly higher in GroupⅡthan in GroupⅠ(P<0．05) ,and thepercentage of CD8 cells was significantly lower in GroupⅡthan in GroupⅠ(P<0．05) .But the difference of percentage of CD3, CD4 and CD8 among GroupⅠ,Ⅲ,Ⅳand V was not significant.The ratio of CD4/CD8 in spleen was 1．6±1．1 in GroupⅠ, 6．2±2．3 in GroupⅡ,4．7±1．7 in GroupⅢ, 3．5±2．1 in GroupⅣand 1．8±0．7 in GroupⅤ. The ratios inGroupⅡ,ⅢandⅣwere significantly higher than that in GroupⅠ(P<0．05) .The ratioin GroupⅤwas slightly higher than that in GroupⅠ, but not significantly.The ratio of CD4/CD8 in peripheral lymphoid nodes was 9．2±4．3 in GroupⅠ,17．1±5．8 in GroupⅡ, 13．4±5．3 in GroupⅢ, 12．9±7．2 in GroupⅣ, 11．2±6．1 in GroupⅤ. The ratios in GroupⅡ,ⅢandⅣwere higher than that in GroupⅠ(P<0．05) . Theratio in GroupⅤwas slightly higher than that in GroupⅠ, but not significantly.The pathology assessment scores were 1．40±0．32 in GroupⅠ, 3．00±0．20 in GroupⅡ, 2．30±0．24 in GroupⅢ, 2．10±0．36 in GroupⅣ, and 1．70±0．24 in GroupⅤ. Thescores in GroupⅡ,ⅢandⅣwere significantly higher than that in GroupⅠ(P<0．05 ).The score in GroupⅤwas slightly higher than that in GroupⅠ, but not significantly.Conclusion EPC donor heart perfusion before implantation with CsA feeding aftertransplantation could adjust the T lymphocytes ratio and ameliorate the acute rejectionin allograft heart.Part 7．The Expression of Cytokines in Allograft Perfused with EPCsafter heart transplantationObjective To study the mechanism of immune regulatory effect of EPCs perfusion bycytokine expression.Methods Twenty pairs of Wistar rats and SD rats were randomizely divided into fourgroups (5 pairs in each group). The animal transplantation model in Part 3 was used.GroupⅠwas set up as the control. GroupⅡ, the rats were fed with CsA aftertransplantation for 7 days. GroupⅢ, the donor heart were perfused with 1×10~6 EPCsbefore implantation. GroupⅣ, the donor hearts were perfused with 1×10~6 EPCsbefore implantation and the rats were fed with CsA for 7 days after transplantation.On the seventh day after transplantation, allograft hearts were extracted for real-time quantity PCR for the tissue RNA expression of INF-γ, IL-2, TNF-α, TGF-β, IL-10and VEGF.Results The tissue RNA expression of INF-γ,IL-2 in GroupⅡ,ⅢandⅣwere lowerthan those in GroupⅠ(P<0．05) .There was no difference of IL-8 expression amongfour groups. The expression of TGF-βin GroupⅡandⅣwere higher than that inGroupⅠ(P<0．05) . The expression of TNF-αin GroupⅡ,ⅢandⅣwere lower thanthat in GroupⅠ(P<0．05) . The expression of VEGF in GroupⅢwas the highest, whileGroupⅣwas the lowest among four groups.Conclusion Donor heart perfusion with EPCs before implantation plus CsA feedingafter transplantation could ameliorate the acute rejection in allograft heart by cytokinepathway.