| BackgroundWith the development of modern medicine,tissue and organ transplantation has become an important treatment method for many diseases.Although the application of immunosuppressive agents can prolong the survival time of allograft,the possible side effects of immunosuppressive agents and the risk of opportunistic infection still limit the application of immunosuppressive agents,and lifelong medication also causes great burden to patients.Therefore,to find a method that can induce immune tolerance of allograft,increase the survival time of the graft with low toxicity and good safety is an urgent problem to be solved in transplantation technology.Mesenchymal Stem cells(MSC),which was originally discovered in the bone marrow by Friedenstein in 1976,is a kind of adult stem cells capable of self-renewal and multiple lineage differentiation.MSC exists in all kinds of tissue in the body,especially the interstitial connective tissue and organs,such as skeletal muscle,fat,bone marrow,placenta,and even in the peripheral blood,among which bone marrow has the highest concentration.Bone Mesenchymal Stem Cell(BMSC)has a fibroblast-like morphology in vitro,showing a vortex-like adherent growth.Under specific culture conditions,they can be induced to differentiate into a variety of tissue cells,such as osteoblasts,adipocytes and chondrocytes,and even into cardiomyocytes,lung epithelial cells,liver cells and nerve cells,etc..BMSCS can express a variety of surface antigens,such as SH2,SH3/CD29,CD44,CD75,CD90,CD105,etc.and express of CD14,CD34 and CD45 at low level,however,specific markers of BMSC has not been found yet.At the same time,BMSC express low level of majorhistocompatibility complex-I(MHC-I)molecules,and do not express of MHC-II class molecules,as well as B7-1,B7-2,CD40,CD40L,CD80 and other necessary co-activated molecules for the activation of T cells,which makes the BMSC has immune exemption,and greatly expand its clinical application.At the same time,BMSC can regulate a variety of immune cells by directly contacting with a variety of immune cells or indirectly acting on a variety of immune cells by paracrine of a variety of soluble cytokines,thus playing a role in immune regulation.These immunological characteristics of BMSCS have attracted much attention and research in the clinical application of tissue and organ transplantation,tissue injury repair and the treatment of autoimmune diseases.Transforming growth factor-?(TGF-?)subfamilies include at least six subtypes(TGF-? 1-6),and only three subtypes(TGF-?1,TGF-(32,TGF-?3)are expressed in mammalian.Among them,TGF-?1 widely exists in various kinds of normal cells transformed cells in the body,which has extensive biological effect,can affect fibroblasts,stimulating their proliferation and differentiation,and participate in the formation of extracellular matrix(ECM),promote the vascular endothelial cell proliferation,is conducive to the formation of blood vessels,etc.,involved in wound healing,immune response,tumor development,fibrosis,and so on many physiological processes.In addition,TGF-?1 can inhibit the activation and proliferation of effector T cells,inhibit the differentiation of T lymphocytes into CTL,interfere with the differentiation of CD4+T cells into Thl cells,inhibit the activation and proliferation of B cells,as well as the activation of macrophages and NKC,and up-regulate the expression of Treg cells,with a strong immunosuppressive effect.With the development of research,BMSC have been found to be useful in the treatment of many diseases.Early application of BMSC in graft-versus-host disease(GVHD)patients can significantly improve the one-year survival rate.BMSC can induce graft immune tolerance,effectively protect the graft,improve the graft function and prolong the graft survival time in renal transplantation,liver transplantation,skin transplantation and other tissue and organ transplantation patients.In patients with myocardial ischemia,spinocerebellar ataxia(SCA),liver cirrhosis and other diseases,BMSCS can significantly improve the function of damaged organs,thereby improving the clinical symptoms of patients and alleviating the pain causedby the disease.BMSCS have also shown efficacy in patients with autoimmune diseases such as systemic lupus erythematosus(SLE)and rheumatoid arthritis(RA).Studies on the application of TGF-? in patients with tissue and organ transplantation have also found that it can significantly inhibit the inflammatory response caused by early acute rejection,reduce the damage of rejection to the graft,and significantly extend the survival time of the graft.However,it has also been found that TGF-? can lead to fibrosis and vascular lesions of the graft in the later stage of graft rejection,thus affecting the survival time and quality of life of patients,which significantly limits the future clinical application of TGF-?.Both BMSC and TGF-?,which have been recognized as immunosuppressive factors,have a strong effect on the induction of graft immune tolerance,which makes us wonder about the effect of the combination of the two.In order to explore the effect of BMSC and TGF-? combined on immune regulation,this research studied it in vitro and in vivo,and further explored the mechanism of its occurrence by detecting relevant indicators,so as to provide new ideas for clinically available BMSC treatment schemes to induce graft immune tolerance.ObjectiveThe effect of BMSC and TGF-? combined with BMSC on the induction of graft immune tolerance was explored in vitro and in vivo,and the mechanism of its occurrence was further studied through the detection of relevant indicators,so as to provide new ideas for clinical BMSC treatment schemes for the induction of graft immune tolerance.At the same time,in view of the possible adverse consequences of direct application of TGF-?,pretreatment of BMSC with TGF-? was used as a specific means of combination of the two.Skin transplantation models were easier to obtain and easier to observe compared with other transplantation models,so it was selected as the experimental model for in vivo study.Methods1.Femur and tibia of Balb/c mice were selected to extract cells by adherent culture method,and cultured and subcultured in vitro.The extracted cells were identified as BMSC by observing their morphology,detecting their surface markers by flow cytometry,and detecting their ability to differentiate into osteoblasts and lipids by inducing differentiation in different directions.2.Different proportions of BMSC were co-cultured with T lymphocytes stimulated by concanavalin A(ConA),and the proliferation of T lymphocytes was detected by MTT assay.BMSC were pretreated with different concentrations of TGF-? and then co-cultured with Cona-stimulated T lymphocytes.The proliferation of T lymphocytes was detected by MTT assay.3.A skin transplantation model was established using Balb/c mice as the donor and C57BL/6 mice as the recipient.4.BMSC and BMSC pretreated with TGF-?(BMSC-TGF-?)were labeled with CM-Dil and injected into skin graft model recipient mice via tail vein.Skin graft samples were collected 7 days after skin transplantation to observe whether they migrated to the graft wound.5.BMSC and BMSC-TGF-? were injected into the skin transplantation model mice via tail vein to observe the survival time of transplanted flap.At different time after transplantation,the transplant flaps were collected and observed under an electron microscope,spleen samples of the mice were collected and then analyzed by flow cytometry to detect the proliferation of CD4+CD25+Foxp3+Treg,and mice peripheral blood was drawed and then analyzed by ELISA to detect the expression level of IL-10,TGF-?,IL-2,and IFN-y.Results1.BMSC was successfully extracted from the femur and tibia of Balb/c mice by adherent culture,which presented typical fibroblast-like morphology and characteristic whirlpool growth under the microscope.Flow cytometry was used to detect the phenotype,and expression of CD44 and CD90 and negative expression of CD14 and CD45 was confirmed.The extracted cells were successfully induced to differentiate into osteoblasts and adipocytes,confirming that they were BMSC.2.In vitro,BMSC can significantly inhibit the proliferation of T lymphocytes induced by ConA,and the inhibitory effect is proportional to the BMSC concentration.BMSC pretreated with different concentrations of TGF-? still significantly inhibited the proliferation of T lymphocytes induced by ConA stimulation,but the inhibition was partially reversed compared with unpretreated BMSC.3.BMSC and BMSC-TGF-? labeled with CM-Dil were observed on the graft wound surface of the skin sample taken 7 days after tail vein injection into recipient mice.4.In the mouse skin transplantation model,the skin grafts of recipient mice in the blank control group showed extensive scab formation or necrosis and exfoliation earlier,and the survival time was only 8.011.05 days.The survival time of the graft was significantly prolonged(12.5±1.35 days)in the BMSC group.The survival of the grafts in the BMSC-TGF-p group was between the two groups at 10.6±1.90 days.At 3,7,10 and 14 days after transplantation,it was obvious that the proportion of CD4+CD25+Foxp3+Treg cells in the spleen of recipient mice in BMSC group was higher compared with blank control group(p<0.05).At 3,7 and 14 days after surgery,the proportion of spleen Treg cells in BMSC-TGF-? group was remarkably higher compared with blank control group.Moreover,the proliferation of Treg cells in BMSC group was significantly earlier than that in BMSC-TGF-p group(p<0.05).At 7 and 10 days after transplantation,the expressions of IFN-? and IL-2 were markedly inhibited while the expressions of IL-10 and TGF-? were significantly enhanced in both the BMSC group and the BMSC-TGF-? group compared with the blank control group(p<0.05).14 days after surgery,the inhibitory effect of IL-2 and IFN-?expression and enhancement effect of IL-10 expression of BMSC is still significant(p<0.05),but the expression of TGF-p in turn significantly inhibitory effect(p<0.05),at the same time,BMSC-TGF-p inhibition of the expression of IL-2 and IL-10,TGF-p enhancement effect is still significantly(p<0.05),but the inhibition of the expression of IFN-? has weakened to similar to blank control group(p>0.05).Conclusion1.BMSC was successfully extracted from the femur and tibia of mice,cultured in vitro and subcultured,while maintaining the stability of cell biological characteristics.2.Both BMSC and BMSC-TGF-? could be transplanted into the wound surface of skin graft model mice after tail vein inj ection.3.BMSC revealed strong immunosuppressive effect in vitro and in mouse skin transplantation model.4.TGF-? can partially reverse the immunosuppressive effect of BMSC,both in vitro and in mouse skin graft models.5.The immunosuppressive effect of BMSC and the partial reversal of the immunosuppressive effect of TGF-? on BMSC may be related to the varying degrees of regulation of Treg cells and the expressions of IL-10,TGF-?,IL-2 and IFN-?. |
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