| Background:Acute myocardial infarction(AMI)is a popular ischemic myocardial disease with high mortality.Timely revascularization is helpful for reducing myocardial damage.However,reperfusion of ischemic myocardium can induce varying degrees of myocardial injury.Inappropriate inflammatory response is an important mechanism of myocardial reperfusion injury.Many studies suggest that mesenchymal stem cells(MSCs)have a strong anti-inflammation effects in a severe inflammatory environment through immunsuppressive function.Therefore,from the point of regulating inflammation,the optimal timing of MSC delivery may be the beginning of the reperfusion.The present study was designed to investigate the effect and mechanism of human umbilical cord mesenchymal stem cells(HUCMSCs)transplantation immediately after AMI on improving myocardial injury,promoting myocardial repair and regulating inflammation,which can provide reference for the optimization of stem cell therapy for reperfusion injury of AMI.Methods:1.HUCMSCs were cultured in vitro and labeled with Ferumoxytol-PLL.Flow cytometry was used to identify cell surface markers.2.After the left anterior descending branch was blocked 90 minutes by coronary artery balloon,blood flow was restored to establish an AMI reperfusion model.3.Experiment grouping:Experimental Chinese minipigs were divided into three groups:(1)Control group:PBS was injected via coronary artery immediately after reperfusion of AMI;(2)HUCMSC group:injection of 5x10~7 HUCMSCs via coronary artery immediately after reperfusion of AMI;(3)HUCMSC+Cs A group:Immediately after reperfusion of AMI,5x10~7HUCMSCs were injected via coronary artery and Cs A infused intravenously 20 minutes before cell delivery;4.CMR imaging was performed before AMI,24h,1 week and 2 weeks after transplantation,and T2*observe the homing of transplanted cells;first pass myocardial perfusion imaging was performed to observe regional myocardial perfusion;delayed enhanced observation was given for infarcted myocardial area and MVO;cardiac movie imaging for local cardiac function;5.Animals were put to death 2 weeks later,and infarct core areas,infarct surrounding areas,and normal areas were embedded and fixed to make paraffin sections.Prussian blue staining was used to observe the distribution of iron-containing cells;Masson staining to detect local myocardial collagen deposition;Ki67 immunohistochemistry was tested to observe cell proliferation;v WF immunohistochemistry to observe angiogenesis;Caspase3 immunohistochemistry to detect apoptosis;CD68 immunohistochemistry to detect local myocardial macrophage infiltration;CD3 immunohistochemical detection for T cell infiltration;TUNEL and CD3 immunofluorescence double staining detection for T cell apoptosis;Ki67 and CD3 immunofluorescence double staining to detect T cell proliferation;PBMC were extracted at different time points(before modeling,24h,4 days,7 days,14 days after reperfusion)from peripheral blood,and the ratio of CD4+CD25+foxp3+Treg cells to CD4+T cells was measured by flow cytometry.Results:1.It was found no change in surface markers of HUCMSCs after subculture to P6,with high expression of CD73,CD90,CD105,CD44,low expression of CD19,CD11b,CD34,CD45,HLA-DR,HLA-DQ.2.Firstly,the difference between the control group and the HUCMSC group was compared.In vivo CMR experiments,delayed enhanced imaging showed that the infarct volume was smaller in HUCMSC group than in control group;the LVEF of two groups before surgery was of no significant difference,and was decreased in both groups after surgery,but no significance;as shown by the first pass myocardium perfusion,in HUCMSC group,after 24 hours of transplantation,the perfusion value of myocardial infarction core area was lower than that of the control group(P<0.01).After one week,the HUCMSC group was no different from the control group;no obvious T*2WI low signal at any of the three time points in HUCMSC group;USPIO blue-stained particles in Prussian blue-staining were detected in HUCMSC group;area ratio of collagen deposition in HUCMSC group was lower than that of the control group(p<0.05);the density of neovascular in the infarct core area and infarct surrounding area of the HUCMSC group was higher than that of the control group(p<0.001);Ki67 test showed the number of proliferating cells in the infarct zone and the periphery zone in HUCMSC group was higher than control group(p<0.001);Caspase3 test showed that the apoptosis rate of the control group in the infarct area and the periphery area was higher than that of the HUCMSC group(p<0.001);the number of CD3 positive T cells in the infarct core area of the HUCMSC group was significantly more than that in control group,while the CD68 positive macrophages of the control group in the infarct core area was significantly more than HUCMSC group(p<0.001);the apoptosis rate of CD3 positive T cells in HUCMSC group was significantly lower than that in control group;Foxp3 positive cells in the infarct core area of HUCMSC group was significantly more than that in the control group(p<0.05);The proportion of CD4+CD25+foxp3+Treg cells in the peripheral blood of the control group in the CD4+T cells increased significantly after 4 days upon the operation and was significantly higher than that in the HUCMSC group and HUCMSC+Cs A group(p<0.05).These results showed that immediate delivery of HUCMSCs via coronary reduced reperfusion injury after AMI.3.In this part,the difference between the HUCMSC+Cs A group and the HUCMSC group was compared.The infarct volume was higher in HUCMSC+Cs A group than in HUCMSC group at each detection time points(P<0.05);the LVEF was decreased significantly in HUCMSC+Cs A group at any of the three time points after surgery(P<0.001);the perfusion value of myocardial infarction core area in HUCMSC+Cs A group was lower at any of the three time points after surgery than that in HUCMSC group;USPIO blue-stained particles in Prussian blue-staining of the HUCMSC+Cs A group were lower than that of the HUCMSC group(P<0.01);the area ratio of collagen deposition in HUCMSC+Cs A group was higher than that in HUCMSC group(p<0.05);the density of neovascular in the infarct core area and infarct surrounding area of the HUCMSC+Cs A group was less than that of the HUCMSC group;Ki67 test showed the number of proliferating cells in the infarct zone and the periphery zone in the HUCMSC+Cs A group was less than the HUCMSC group(p<0.001);the number of CD3 positive T cells in the infarct core area of HUCMSC+Cs A group was less than that in HUCMSC group(p<0.001);the apoptosis rate of CD3 positive T cells in HUCMSC+Cs A group was significantly higher than that in HUCMSC group(p<0.001);Foxp3 positive cells in the infarct core area of HUCMSC+Cs A group was less than that in the HUCMSC group(p<0.05).The results showed that immunosuppressive agent reversed the protective effect of immediate coronary transplantation of HUCMSCs after AMI reperfusion by the inhibition of T cells and inflammatory response.Conclusion:Immediate transplantation of HUCMSCs by coronary perfusion after AMI is an optimized treatment for reducing myocardial damage and promoting myocardial repair.This was mainly achieved through the regulatory of inflammatory by reciprocation with T lymphocytes. |
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