| PurposeUsing genetic engineering techniques to overexpress programmed cell death 1 ligand 1protein on macrophage membranes and preferentially select macrophage subtypes to overexpress β2 integrins and multiple inflammatory factor receptors.To construct overexpressed programmed cell death 1 ligand 1 cell membrane-coated bionanoparticles and validate the physiological activities of their effector molecules including programmed cell death1 ligand 1,β2 integrin,and inflammatory factor(IL-1β,IFN-γ,IL-6,TNF-α,MCP-1)receptors.To verify the ability of cell membrane-coated bionanoparticles to modulate the T-OF co-culture system and the therapeutic effect in thyroid associated ophthalmopathy animal models,and to explore a thyroid associated ophthalmopathy therapeutic strategy that focuses on building immune tolerance.Menthods1.Construction of cell lines overexpressing programmed cell death 1 ligand 1,β2 integrin and inflammatory factor receptor: programmed cell death 1 ligand 1 overexpression and knockdown strains were constructed using genetic engineering techniques,and subtypes of THP-1 were selected with high expression of programmed cell death 1 ligand 1,β2 integrin and inflammatory factor receptor.2.Construction of cell membrane-coated bionanoparticles and phenotype identification:Cell membrane proteins were extracted and coated on the surface of Poly lactic-co-glycolic acid nanoparticles,and the stability and safety of cell membrane coated bionanoparticles were examined.3.Inhibitory effect of cell membrane-coated nanoparticles on T-cell activity: Peripheral blood T cells from thyroid associated ophthalmopathy patients were extracted and activated for amplification,and the effects of programmed cell death 1 ligand 1 on T cell apoptosis,Treg subpopulation differentiation,T cell CD40 L expression and secretion of inflammatory factors were investigated.4.Adhesion of cell membrane coated nanoparticles to OF cells and adsorption of inflammatory factors: Primary OF from thyroid associated ophthalmopathy patients and control group were used to verify the high expression of ICAM-1 in OF of thyroid associated ophthalmopathy patients,and the adhesion and adsorption of inflammatory factors of cell membrane coated nanoparticles were detected.5.Regulation of OF-T cell co-culture system by cell membrane-coated nanoparticles: The ability of cell membrane-coated nanoparticles to regulate the level of inflammatory factors in the OF-T cell co-culture system,to inhibit the level of HA secretion by OF cells,and to affect the MAPK and NF-κB p65 signaling pathways in OF cells were investigated.6.Therapeutic effects of cell membrane-coated nanoparticles on thyroid associated ophthalmopathy animal model: The therapeutic effects were evaluated by staining of orbital soft tissue sections,inflammatory factor microarray and small animal nuclear magnetic imaging after treatment with cell membrane-coated nanoparticles.Results1.The m RNA transcript levels of programmed cell death 1 ligand 1 were successfully modulated in the overexpression and knockdown strains,as demonstrated by q PCR.Western blot and flow cytometry confirmed the protein expression levels of programmed cell death 1ligand 1 were similarly affected in the overexpression and knockdown strains.After differentiating THP-1 into M0,M1 and M2 isoforms,the M1 isoform was found to have high expression of the three effector proteins,and was selected for subsequent experiments.2.The translucent cell membrane wrapped around the core of Poly lactic-co-glycolic acid nanoparticles can be visualized by transmission electron microscopy.The successful construction of cell membrane-coated bionanoparticles was also demonstrated by measuring the particle size and potential of cell membrane-coated nanoparticles by laser particle size measurement.The cell membrane-coated bionanoparticles maintained a stable particle size in aqueous solution for 6 days,demonstrating the cell membrane-coated bionanoparticle stability.3.Programmed cell death 1 ligand 1 on cell membrane-coated nanoparticles was found to induce T cell apoptosis,Treg subpopulation differentiation,and inhibit T cell expression of CD40 L and secretion of inflammatory factors(TNF-α,IFN-γ,IL-6),using programmed cell death 1 ligand 1 overexpression and knockdown strains to prepare the nanoparticles.It was demonstrated that programmed cell death 1 ligand 1 on the surface of cell membraneencapsulated nanoparticles could induce T cells to change from a pro-inflammatory phenotype to an anti-inflammatory phenotype.4.The expression of ICAM-1 on OF in thyroid associated ophthalmopathy patients was verified to be higher than that in healthy subjects,which could build specific immune tolerance against OF through β2 integrin-mediated adhesion to cell membrane-coated nanoparticles.Within 8 hours,the cell membrane-coated nanoparticles stably adhered to the surface of orbital fibroblasts and were not phagocytosed by orbital fibroblasts.Cell membrane-coated nanoparticles were also found to adsorb inflammatory factors through inflammatory factor receptors on their surface.Significant dose-dependent effects between added cell-coated nanoparticles and residual inflammatory factors in solution.5.The cell membrane-coated nanoparticles reduced the concentration of various inflammatory factors in the OF-T cell co-culture system,inhibited the ability of OF cells to secrete HA levels,and reduced the phosphorylation of MAPK pathway(p38,ERK1/2,JNK)in OF cells.It was demonstrated that the cell membrane-coated nanoparticles could comprehensively regulate the inflammation level of OF-T cell co-culture system,inhibit the abnormal activation of OF cells,and delay the pathogenic effect of OF cells.6.Repeated stimulation of mice with oxazolone semi-antigens successfully induced T cellmediated predominant autoimmune inflammation in the orbital region.Treatment with cell membrane-coated nanoparticles in the thyroid associated ophthalmopathy animal model resulted in reduced inflammatory edema of orbital soft tissues,decreased levels of inflammatory factors,and reduced infiltration of inflammatory cells,demonstrating the therapeutic effects of these nanoparticles.This indicates their potential use as a thyroid associated ophthalmopathy therapeutic strategy based on the construction of immune tolerance.ConclusionsIn this project,we preferentially selected macrophage subtypes and expressed programmed cell death 1 ligand 1,β2 integrin,and inflammatory factor(IL-1β,TNF-α,IFN-γ,IL-6,MCP-1)receptors on the cell membrane surface.We successfully constructed cell membrane-coated bionanoparticles overexpressing programmed cell death 1 ligand 1,which can specifically target the surface of orbital fibroblasts(OF)by expressing programmed cell death 1 ligand 1 and inflammatory factor receptors.This approach can inhibit the activation of OF,regulate the immune microenvironment,reduce hyaluronic acid deposition,and alleviate the pathological process of thyroid associated ophthalmopathy animal models.This study offers a new strategy and application method for thyroid associated ophthalmopathy treatment,as it has the potential to reshape orbital immune tolerance. |
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