| Background and objective:Diabetes is a group of metabolic diseases characterized by hyperglycemia.The long-standing hyperglycemia in patients with diabetes can lead to chronic damage and dysfunction of eye,kidney,heart,nerve and other organs.Clinically,multiple injections or continuous subcutaneous insulin injections can delay or slow the occurrence or progression of chronic complications of diabetes.However,despite advances in insulin preparations,insulin delivery devices and capillary blood glucose monitoring,some patients are still unable to achieve relatively safe metabolic control,resulting in recurrent hyperglycemia or hypoglycemia,even life-threatening.Islet transplantation is an effective treatment for diabetic patients whose blood glucose level is difficult to be controlled by exogenous insulin.Islet transplantation supplements the deficiency of endogenous insulin levels by injecting donor islets into patients with diabetes.Clinically,the preferred surgical method is portal vein injection.More than 90% of islet transplantation is performed by this method.Islet cells are infused into the liver.Islet cells grow in the liver sinus and secrete insulin.The renal capsule,spleen,peritoneum,and omentum are also important transplant sites.After the Edmonton scheme was promoted,the insulin detachment rate could reach 50% after 5 years of transplantation,suggesting that current islet transplantation still has some limitations.The early loss of islet cells due to rejection and the adverse drug reactions of immunosuppressants are still the main reasons for the poor long-term effect of islet transplantation.The occurrence of T cell-mediated rejection after islet transplantation is an important factor in the loss of islet allografts.Therefore,to further study the main mechanism of rejection after islet transplantation and find new means of anti-rejection is an urgent hot issue to be solved in the basic research of islet transplantation.Immune checkpoints are a series of inhibitory signaling pathways that regulate the immune system in the body,which are of great significance for the body to regulate immune response,maintain immune tolerance and prevent tissue damage.The activation and high expression of immune checkpoints can inhibit the proliferation and function of activated T cells,down-regulate the activity of T cells,and play a balanced role in the regulation of human immune system.In tumor immunotherapy,the anti-tumor effect is exerted by blocking the receptor-ligand interaction at the immune checkpoint and reversing the immune depletion state of T cells.In this study,the expression of immune checkpoint molecules in allogeneic islet transplantation in mice was analyzed to screen the targets with potential anti-rejection effect.At the same time,a stable liposome surface modification system was constructed to mount target ligand molecules and prolong the in vivo half-life of immune checkpoint ligand molecules in order to inhibit lymphocyte activation induced by alloantigen in vivo.Finally,the protective effect of the ligand-conjugated liposome on islet allografts was observed,and the mechanism of its inhibition of rejection was studied,so as to explore a new therapeutic strategy for early anti-rejection of islet transplantation.Part Ⅰ: Analysis of the expression of immune checkpoint molecules in the process of islet allograft rejection.Objective: To study the expression changes of immune checkpoint receptor proteins during islet allograft rejection and to screen potential anti-rejection targets.Methods: The expression of immune checkpoint receptor protein was detected by Western-Blot through mixed lymphocyte culture of allogeneic mice and activation and expansion of T cells in vitro.Results: During mixed lymphocyte culture and T cell activation and expansion in vitro,VISTA protein was expressed throughout the activation process,LAG-3 protein was mainly overexpressed in the early stage of activation,and PD-1 protein was overexpressed in the late stage of activation.Conclusion: In the process of rejection of the same islet graft,the combined targeting of immune checkpoint receptors LAG-3 and PD1 or the targeting of VISTA alone may have potential anti-rejection effects.Part Ⅱ: Establishment of liposome surface modification system.Objective: To establish a liposome surface modification system for further stable construction of artificial liposomes loaded with immune checkpoint ligand molecules.Methods: 1.Construction of liposomes by ultrasonic thin-film method,the use of streptavidin-biotin system to mount related protein elements;2.Immunofluorescence detection of liposome mounting efficiency.Results: The diameter of artificial liposomes prepared by this method was stable between 100-200 nm,and biotinylated protein fragments could be mounted.The artificial liposome system has higher mounting efficiency and better mounting specificity at the same time.Conclusion: This method of liposome surface modification system is stable and can effectively load immune checkpoint ligands.Part Ⅲ:Preliminary study on the effect of VISTA-conjugated liposome on islet allograft rejection.Objective: To explore the effect of activating immune checkpoint molecules on rejection in islet transplantation with VISTA molecules as the target,and to clarify the necessity of loading liposomes.Methods: 1.To establish an allogeneic mouse mixed lymphocyte culture model and compare the effects of VISTA protein on lymphocyte proliferation and activation;2.The model of islet allotransplantation in mice was established,and the protective effects of different types of drugs on islet allografts were compared and analyzed by setting up normal saline group,10 pmol VISTA protein group,100 pmol VISTA protein group and 100 pmol VISTA-Fc protein group.3.To construct VISTA-conjugated liposome and explore its pharmacokinetic characteristics in vivo.4.Empty liposome group,VISTA liposome group and low dose rapamycin group were set up to study the protective effect of VISTA-conjugated liposome on islet allografts.Results: 1.Compared with the control group,VISTA protein could significantly inhibit the proliferation and activation of lymphocytes.2.Compared with the control group,changing the concentration of VISTA alone could not significantly inhibit transplant rejection.3.Compared with VISTA protein alone,VISTA liposome can significantly prolong the half-life of VISTA protein in vivo.4.Compared with the control group,VISTA liposome could significantly prolong the survival time of islet allografts.Conclusion: Liposome system is helpful for immune checkpoint ligands to play a better biological role in vivo,and it is necessary to load liposomes.Part Ⅳ: Study on the mechanism of FGL1/PDL1-conjugated liposome on islet allograft rejection.Objective: To study the protective effect of FGL1/PDL1-conjugated liposome on islet grafts and to explore the mechanism of their participation in transplantation rejection.Methods: 1.Using the ultrasonic membrane method and streptavidin-biotin system to prepare FGL1/PDL1-conjugated liposome;2.The effect of FGL1/PD-L1 liposome on mixed lymphocyte reaction and the changes of related signal pathways were detected in vitro;3.The model of allogeneic islet transplantation in mice was established,and the mice were divided into normal saline group,FGL1-conjugated liposome group,PD-L1-conjugated liposome group,FGL1/PD-L1-conjugated liposome group and low dose rapamycin group,and the changes of blood glucose and C-peptide levels in mice were regularly detected.Results: 1.FGL1/PDL1-conjugated liposome could significantly inhibit the proliferation and activation of lymphocytes and reduce the secretion of IFN-γ in mixed lymphocyte reaction.PD-L1 inhibits the activation of SYK and VAV mainly by activating SHP1.FGL1 upregulates the expression of TOX and NR4 A.After TOX and NR4 A enter the nucleus,they inhibit the secretion of cytokines,inhibit T cell proliferation and cytotoxic activity,and induce the up-regulation of PD-1 expression to further play an inhibitory role;2.Compared with the other four groups,FGL1/PD-L1-conjugated liposomes could significantly protect islet grafts and prolong their survival time.Conclusion: FGL1/PDL1-conjugated liposome can effectively inhibit transplantation rejection and prolong the survival of islet grafts. |
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