A Comparative Study Of Autologous And Allogeneic Adipose Derived Mesenchymal Stem Cell Transplantation In Acute Myocardial Infarction Rats

The therapeutic effect of mesenchymal stem cells on ischemic cardiomyopathy has attracted wide attention from researchers not only because of its ability to differentiate into cardiomyocytes and vascular endothelial cells,but more importantly,MSC plays an important role in immune regulation in vivo.MSC can not only promote the secretion of anti-inflammatory factors,but also inhibit the expression of pro-inflammatory factors,play a positive role in immune regulation.Bone marrow mesenchymal stem cells have been widely used in the treatment of ischemic heart disease in recent years and have achieved certain efficacy.Compared with BMSCs,adipose mesenchymal stem cells have less damage and more cell proliferation in the process of their acquisition.Strong,rich in MSC content,adipose tissue and ADMSC can also be built for long-term storage,so it has become a hot topic.In addition,allogeneic ADMSCs have weaker immunoreactivity than allogeneic BMSCs.Although many studies have directly compared the immunoreactivity of allogeneic BMSCs and autologous BMSCs in vitro and in vivo,the difference in the efficacy of autologous and allogeneic ADMSCs in the treatment of ischemic animal models has not been directly compared.Therefore,in this study,we extracted autologous and allogeneic adipose-derived mesenchymal stem cells,cultured and expanded and labeled with CM-Di(40)dye,and transplanted labeled cells into acute myocardial infarction rat model.The superiority of adipose-derived mesenchymal stem cells transplantation is expected to establish a safer and more effective treatment method for myocardial ischemic diseases through cell transplantation,which will provide theoretical support for the clinical treatment of related diseases in the future.Part I:Isolation,Cultivation and Identification of Adipose-derived Mesenchymal StemCells from Lewis and BN RatsObjective Adipose tissue was isolated from the abdomen of Lewis and BN rats and the mesenchymal stem cells were cultured and identified.The cells were labeled with CM-DiI and the positive rate was detected.Methods The aseptically collected adipose tissue from the inguinal region of LEWIS and NB rats was mechanically sheared and then digested with collagenase.The isolated cells were fixed in serum-free medium to obtain adipose-derived mesenchymal stem cells.Adipose-derived mesenchymal stem cells which were expanded to passage 3 were induced to differentiate into adipocytes,bone and chondrocytes using flow cytometry.Flow cytometry was used to detect the expression of cell-surface mesenchymal stem cells,and labeled with CM-Dil dye Flow cytometry positive rate of markers,for the next transplant experiments to provide effective cell support.Results Adipose-derived mesenchymal stem cells of the third generation Lewis and BN rats were fibroblast-like and could differentiate into adipocytes,bone and chondrocytes in vitro.The cell surface antigens were detected by flow cytometry.The percentages of CD90 and CD29 on the surface of adipose-derived mesenchymal stem cells derived from the two rats were not lower than 95%.The hematopoietic cell surface markers CD34 and CD45 were not higher than 5%.Flow cytometry to detect the expression of major histocompatibility antigens on the cell surface showed that ADMSCs derived from Lewis and BN rats could express MHC class I molecules(39.82%and 20.97%)higher and MHC-Ⅱ(4.01%and 0.69%).The positive rate of CM-Dil labeled third-generation adipose-derived mesenchymal stem cells was99%.Conclusions Adipose-derived mesenchymal stem cells derived from both rats can be stably passaged,expanded and successfully differentiated into adipose,bone and chondrocytes in vitro.Flow cytometry was used to detect the third generation of ADSCs in line with the International Association of Cell Therapy Mesenchymal stem cell standard.CM-Dil dye labeling rate of fat-derived mesenchymal stem cells positive rate of more than 99%,for the next cell transplantation to make adequate preparations.PartⅡA comparative study of autologous and allogeneic adipose derived mesenchymal stem cell transplantation in acute myocardial infarction ratsObjective Preparation of rat myocardial infarction model,based on the first part of the cell experiments,the cultured rat adipose-derived mesenchymal stem cells were transplanted into myocardial infarction rats,by autopsy and echocardiography compared with autologous adipose-derived mesenchymal stem cells Therapeutic effect of acute myocardial infarction in rats.Methods The model of myocardial infarction was made by Olivette model.Two hours after the model was established,2×10~6 ADMSCs labeled with CM-Dil were implanted into the myocardium of rats.After 7 days of transplantation,Immunofluorescence staining of frozen section of tissue was used to observe the infiltration of CD4+T lymphocytes,CD8+T and CD68 around autologous or allogeneic cell transplantation group.The pathological sections were observed under fluorescent microscopy 28 days after transplantation and compared with the survival of autologous and allogeneic ADMSC,Differentiation and collagen deposition between the two treatment groups.Echocardiography was used to determine and compare the cardiac function of rats in each group.Results Transplantation of adipose-derived mesenchymal stem cells traced by CM-Dil into the myocardium of acute myocardial infarction rats showed that:(1)One week,two weeks,four weeks after cell transplantation,the number of transplanted cells in the periphery of infarcted myocardium was significantly higher than that in autologous ADMSC group Higher than allogeneic ADMSC group(P<0.01).(2)The results of immunofluorescence staining showed that the number of vascular smooth muscle cells and endothelial cells in autologous ADMSCs transplantation group was significantly higher than those in allograft ADMSCs transplantation group.(3)Seven days after the transplantation,the results of immunofluorescence staining showed that CD4+T lymphocytes and CD8+T lymphocytes infiltrated and activated(CD68+)around allogeneic ADMSC,but no infiltration of lymphocytes was found in autologous ADMSC.(4)The results of echocardiography of rats at28 days after transplantation showed that the LVEF index of the two cell transplantation groups was significantly higher than that of the control group,while the LVEDD and LVEDD were decreased(P<0.05).The two cell transplantation groups Compared with allograft ADMSCs transplantation group,LVEF in autologous ADMSCs transplantation group was increased,while LVEDD was decreased(P<0.05).Conclusions In this study,we directly compared the survival,differentiation,immune response and myocardial function of autologous and allogeneic ADMSCs transplanted into the myocardium of acute myocardial infarction model rats,and for the first time confirmed the autologous ADMSC in AMI model rats excellent treatment effect In allogeneic ADMSC.This may be a certain degree of immunogenicity with rat ADMSC,after myocardial transplantation induced immune rejection,is not conducive to the survival of allogeneic ADMSC.In addition,4 weeks after transplantation,autologous ADMSC cells showed significantly more survival than allogeneic ADMSC.Transplantation of autologous ADMSC in addition to playing the same role as paracrine growth factor in allogeneic ADMSC also play a role in cell replacement to further clarify the advantages of autologous ADMSC treatment of AMI model rats for the clinical treatment of myocardial ischemic disease A certain theoretical basis.