| BACKGROUND:With advances in human medical technology and knowledge of new areas,many previously clinically intractable diseases are being solved one by one.Since the success of Barnard's innovative human heart transplantation in 1967,there have been progressive advances in surgical techniques,devices and perioperative patient management.In 1978,Dr.Zhang Shize performed the first heart transplant in China at Ruijin Hospital in Shanghai.After that,heart transplantation in China was gradually carried out.However,with the continuous improvement of people's social living standards,cardiovascular diseases have become a major problem threatening the quality of life and life rights of human beings.For severe end-stage cardiovascular diseases that cannot be improved by medical treatment,these patients are often accompanied by many pathological signs brought about by heart failure,and heart transplantation has become the only means to improve the quality and prolong the survival of patients.Although heart transplantation is currently used as the standard of care for heart failure,post-transplant rejection and the increasing scarcity of graft sources make heart transplantation an urgent medical challenge.With the number of patients with end-stage cardiovascular disease increasing each year and the shortage of available donor hearts for transplantation becoming increasingly critical,the most effective way to address the imbalance in the number of donors and recipients,excluding ethical and social acceptance of organ donation,is to investigate novel ways to induce immune tolerance in recipients,thereby reducing the requirements for graft selection and increasing the utilization of the limited number of donors.This will reduce the requirements for graft selection,increase the utilization of the limited graft donors,and extend the effective survival time of the graft in the recipient to avoid and reduce the impaired graft function due to rejection and the secondary need for transplantation in the same patient.At this stage,graft rejection still continues to be an important factor preventing the establishment of immune tolerance in cardiac grafts.The ideal treatment would be one that induces allograft-specific tolerance in heart transplant recipients while producing fewer adverse side effects compared to current immunosuppressive therapies.We have therefore directed our research to the immune system of the organism,where dendritic cells are an important link in the immune regulation of the organism,serving as an important bridge between innate and adaptive immunity.Their important role in antigen presentation,cytokine secretion and regulation of the immune system is crucial in all immune responses.In particular,in transplantation immunity,dendritic cells are the dominant factor in the recognition of graft-derived antibodies by the recipient immune system.Since their discovery,dendritic cells have been intensively studied and are now considered to be the most powerful immune cells for antigen presentation.Their morphological structure with multiple dendrites and pseudopods,together with the pleomorphic nature of the cell surface,determines their functional properties for efficient uptake,processing and presentation of antigens.Its morphologically determined large cell contact area provides expression sites and enhances the efficiency of contact information exchange for an extremely rich variety of molecules on the surface,including multiple adhesion factors,as well as multiple cell co-stimulatory molecules and multiple antigen-presenting molecules.Dendritic cells are transformed into mature dendritic cells when activated by antigens or corresponding cytokines or other stimulating factors.After maturation,dendritic cells rapidly increase the expression of various immune-related surface molecules and secrete stimulatory cytokines,transforming them into highly immunostimulatory antigen-presenting cells.At this stage,dendritic cells are considered to be the only antigenpresenting cells capable of activating naive T cells that are not exposed to antigen.In addition,some studies have confirmed that dendritic cells after treatment with drugs or molecular regulation can be transformed in another direction,namely the immune tolerant state.In this state,their ability to regulate T cells and other lymphocytes is reversed,i.e.,the role of each lymphocyte in the immune response is suppressed in many ways,and the immune sensitivity of the body is stabilized and down-regulated.The treatment in this study is to induce the immune tolerance state of dendritic cells and thus reduce the effect of immune rejection.In transplantation immunization,the immune response is mainly presented and mediated by dendritic cell processing.Upon entry of the graft into the recipient,the dendritic cells in the graft bind to T cells in the recipient,and once the recipient T cells recognize the graft antigen,the immune system in the recipient begins to mount a strong immune response against the transplanted allogenic antigen.This is the most critical reason for acute immune rejection.Then,if the immune system of the recipient can be pre-treated with graft-derived dendritic cells in the immune tolerance state before the graft enters the recipient,it may be possible to reduce the immune sensitivity of the recipient to the graft.This idea has been continuously confirmed in studies in recent years.However,most drugor cytokine-based regulatory mechanisms lack potency and specificity,and studies targeting alterations at the molecular level of dendritic cells are emerging,furthering our understanding of the molecular mechanisms underlying dendritic cell growth and development while exploring novel therapeutic targets.Recent evidence suggests that cyclic RNA is a novel gene regulator in cell development.The heart is also one of the organs where cyclic RNA is highly expressed.In our previous experiments,we found that the expression profiles of cyclic RNAs differ greatly between mature and immature dendritic cells(circFSCN1 is one of them),in order to investigate the underlying molecular mechanisms.Our aim was to demonstrate that treatment with Immunosuppressive Dendritic Cells(Im DCs)generated after silencing of circulating RNA FSCN1(circFSCN1)in heart transplantation could prevent allogeneic immune rejection and prolong heart transplantation survival.METHODS AND MATERIALS:In vitro transfection of mouse bone marrow-derived dendritic cells with circFSCN1 siRNA.The expression level of circFSCN1 was determined by designing circular RNA-specific reverse primers for q RTPCR to determine its down-regulation effect.And then flow cytometry was used to determine the specific surface molecules(CD40,CD80.CD83 and MHC II etc.)of dendritic cells to determine their effect on dendritic cell developmental maturation.We also designed biotinylated specific DNA probes and determined the location of circRNA expression in dendritic cells under fluorescence microscopy using RNA in situ hybridization.In addition,to determine whether dendritic cells with down-regulated circFSCN1 expression have a corresponding change in their stimulatory function towards T cells in addition to phenotypic changes,we performed Mixed Lymphocyte Reaction(MLRs)to perform assessment of dendritic cell activation of T cells and production of regulatory T cells(Tregs).In addition,to determine the feasibility of a therapeutic approach for immune-tolerant dendritic cell pretreatment of transplant recipients,this study also performed an allogeneic mouse heart transplantation experiment in which recipients were pretreated with donor-derived circFSCN1-silenced immunosuppressive dendritic cells and underwent heart transplantation on the seventh day after treatment,with low-dose rapamycin administered for a short period of time after surgery,followed by conversion to normal feeding,heartbeat was monitored during the same period to assess immune rejection,and the time of immune rejection onset was calculated by graft heartbeat arrest.After 100 days postoperatively,heart graft samples were collected and fixed and embedded,and HE and Trichrome staining were performed to determine tissue damage and tissue fibrosis.The spleens of recipient mice were also extracted and flow cytometric assays were performed on Treg cell fractions.RESULTS:The exon circular RNA circFSCN1 is expressed in the cytoplasm of dendritic cells.As dendritic cells grow and mature,the level of circFSCN1 expressed in dendritic cells gradually increases.Treatment of dendritic cells with different drugs can cause dendritic cells to stay at the immature dendritic cell stage,while the expression of circFSCN1 in the inhibited state of dendritic cells decreases significantly.Conversely,if dendritic cells are treated with stimulatory drugs,they can be put in the activated state,when the expression of circFSCN1 in dendritic cells increases significantly.Silencing circFSCN1 using siRNA can cause it to remain in an immature state,impairing the ability of dendritic cells to activate T cells,and the immunosuppressive effect of low-reactive T cells obtained by this pathway is specific.Not only that,circFSCN1-silenced dendritic cells significantly enhanced Treg transformation and production in mixed lymphocyte culture.Treatment with circFSCN1-silenced immune-tolerant dendritic cells prevented allogeneic immune rejection,prolonged allograft survival,reduced heart graft injury and fibrosis,and induced regulatory T cells in the recipient.CONCLUSIONS:Knockdown of circFSCN1 induces Tol-DCs and treatment with these Tol-DCs prevents alloimmune rejection and prolongs allograft survival.This is a promising therapeutic target to combat transplant rejection in heart transplantation and increases our understanding of circRNA in the immune system. |
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