| BackgroundThe main cause of common clinical ophthalmocace is corneal blindness, corneal transplantation is the most important and the most effective treatment of irreversible corneal blindness. In recent years, tissue and organ transplantation has achieved great progress and widely used in various treatment. Corneal has the unique characteristics of ocular tissue anatomy, immunology and mechanical barrier. In the absence of risk factors for corneal transplant cases, graft survival reach 90% after operation two years. Corneal immune privilege is not absolute sense of organization, therefore, keratoplasty is difficult to avoid immune rejection. Since the transplant recipient bed planting a large number of angiogenesis and long-term inflammation caused by immune rejection, the surgical success rate has dropped significantly. Therefore, prevention and treatment of corneal transplantation immune rejection, improve corneal graft survival and improve prognosis after surgery is still a hot issue in recent years.In recent years, the most important treatment of corneal allograft rejection include local or systemic corticosteroids and cyclosporine A and other drugs. The above-described method had some satisfactory results in clinical studies, but these immunosuppressive effect of its clinical dosage and time was a significant positive correlation. Because of its non-specific response characteristics, clinical application process will severely damage the immune system mechanisms of the patient. A variety of complications arising exacerbated immune rejection occurs, that leading to the failure of keratoplasty.Dendritic cells are the efficiently antigen presenting cells, it can not sensitized naive T cell proliferation induced activation of T cell immune response while promoting dependent antibody production. Dendritic cells have significant heterogeneity of differentiation, differentiation and maturation process experienced in the various precursor cells. Immature cells and mature cells not only on the cell phenotype differences are significant, their role in the immune response is also in the process of each Are not the same. The precursor cells into immature dendritic cells, located in the body’s peripheral tissues between organs stroma, when the dendritic cells have a strong phagocytic capacity and antigen processing of exogenous antigen and bind to the endogenous tissue phase capacitive on complex molecules. Immature dendritic cells in a stable environment within the body in a resting state, then the migration is non-antigen-dependent, and when the body by stimulating immature dendritic cells with an exogenous antigen-dependent manner in accordance with the migration, and part of the migration process by stimulating factors gradually to mature dendritic cell differentiation, activation and participation start the body’s immune response mechanism in the process. The study presents both immature dendritic cell phenotype and is capable of inducing T cell tolerance tolerogenic dendritic cells called dendritic cells. By inducing bone marrow-derived dendritic cells in vitro to differentiate into tolerogenic dendritic cell-mediated immune tolerance, thereby treating corneal allograft rejection is a practical experimental research program.The stable tolerogenic dendritic cells are key to promote immune tolerance and prolong graft survival. The current method were induced differentiation of immature dendritic cells obtained mainly through granulocyte-macrophage colony-stimulating factor or co-culture of rapamycin, cyclosporine, corticosteroids and other drugs in vivo and in vitro inhibition of dendritic cells differentiation. The method described above made some satisfactory results in experimental studies, but dendritic cells obtained whether the long-term maintenance of immature dendritic cells phenotype and tolerogenic dendritic cells achieve functional features also open to question.NF-κB is the body’s cells in apoptosis, proliferation, differentiation, and immune response in key regulatory transcription factor. There are more than 150 kinds of genes expression regulating by NF-κB. NF-κB maturation of dendritic cells play an important role. Immature dendritic cells in vivo after antigen stimulation by antigen-dependent migration and gradually to mature dendritic cells during migration. The NF-κB transcription start may participate in MHC, CD80 and CD86. Dendritic cells during maturation of MHC, CD80, CD86 and inter-cell adhesion molecules and co stab significantly higher expression, while stimulating naive T cells of the initial immune response. Stimulation of T cell activation induced require dual or multiple signals, these signals including CD80, CD86, MHC antigens and other signals and the corresponding polypeptide mentioned above.NF-κB is a Rel protein and NF-κB family members together constitute the two, is a precursor of NF-κB1 and family, including NF-κB2; the other is the Rel family includes c-Rel, v-Rel, Rel-B and Rel-A (p65). Rel homology domain Rel/NF-κB family includes terminal domain, of DNA binding domain, a dimerization domain and the nuclear localization sequence domain. In addition to having the same among members of homology outside, Rel-A, Rel-B and c-Rel molecule also has a transcriptional activation domain, the functions of the two domains is also somewhat different, transcriptional activation domain is responsible for NF-κB dimers and activate transcription. NLS domains may mediate Rel-A and enter the nucleus by transcription Rel-A, Rel-B, and c-Rel-specific transcriptional activation domain mediates the Rel-A. Rel-A phosphorylation and acetylation is a key influence NF-κB activation and initiation of transcription regulation, the current NF-κB activity regulation research focuses mainly focused on Rel-A protein modification. When serine 276 kinase stimulation by changing the molecular structure of Rel-A under the non-phosphorylated state, enhance binding Rel-A and cAMP response element binding protein, thereby enhancing the transcriptional activity of NF-κB. We hypothesized that the expression of the gene by interfering with Rel-A, affect the degree of phosphorylation of the Rel-A, and then decreased the transcriptional activity of NF-κB to obtain tolerogenic dendritic cells.RNA interference is introducing exogenous and endogenous dsRNA-specific degradation by cells in vivo induction of homologous target gene mRNA sequence, resulting in post-transcriptional gene silencing phenomenon. Since the RNA-induced silencing complex may be utilized and the secondary to catalytic RNA polymerase, so throughout the process and to catalyze RNA amplification effect. siRNA into the development of technology to provide more options for the application of RNA interference technology, siRNA-mediated expression of a variety of viruses is the most effective class carrier. Lentiviral vector is the human immunodeficiency virus type I developed on the basis of gene therapy vectors, although belonging to the category of retrovirus vector. It is used in a broader host to non-dividing cells and dividing cells have the infection, the foreign gene vector can be effectively integrated into the host chromosome, effective against transcriptional silencing so as to achieve the role of persistent expression, can be inserted into the vector specific promoter and enhancer, enhance transgene transcription targeting, so lentiviral vector expression of the gene in a specific tissue cells.The issue on the basis of previous research and the latest developments on the Establishment of rat model of corneal allograft rejection by granulocyte-macrophage colony-stimulating factor and IL-4 co-culture induced differentiation of rat bone marrow-derived dendritic cells, using RNA interference technology, highly efficient at inhibiting siRNA sequence selected Rel-a expression construct for siRNA lentivirus vector cell Rel-a, the lentiviral vector transfected into bone marrow-derived dendritic cells, reducing NF-κB expression and activity, in order to a new method to obtain stable dendritic cells induced tolerance, while the tolerogenic dendritic cells injected into rat corneal transplantation model to explore the Rel-a low expression of tolerogenic dendritic cells induced corneal transplantation immune tolerance and mechanism of action, clinical corneal transplantation immune tolerance induction treatment of immune rejection provide theoretical and experimental basis.Part one. Induced differentiation rat bone marrow-derived dendritic cells and biological characteristic detectionObjectiveCombined with the latest research progress, further explore the in vitro differentiation of immature dendritic cells tolerogenic phenotype and characteristics of dendritic cells.MethodsThis part of the experiment induced bone marrow precursor cells into immature dendritic cells depending on the culture period. Observation on growth and morphology of different culture time of dendritic cells through a microscope; flow cytometry anlysis markers of dendritic cell surface; mixed lymphocyte reaction test verify different culture time of immature dendritic cells induced to differentiate whether the biological characteristics of tolerogenic dendritic cells; enzyme-linked immunosorbent assay IFN-y expression levels of dendritic cells in the culture supernatant.ResultsCD80, CD86 and MHC-II positive expression level of the sixth day cells or eighth day cells significantly lower (p<0.05) than the tenth days cells; In mixed cell response, the absorbance values of the sixth day cells or eight days cells was significantly lower than (p<0.05) the tenth day cells; IFN-y of cell culture supernatant expression levels gradually increased among three groups.ConclusionCo-culture solution of IL-4 at a concentration of 5ng/ml, continuing the culture until the sixth day when harvested immature dendritic cells with a dendritic cell phenotype and having the biological characteristics of tolerogenic dendritic cells. Rat bone marrow dendritic cells to differentiate into tolerogenic dendritic cells during the course of the incubation time and induce differentiation medium precise adjustment is extremely important, a dendritic cell-specific expression of surface markers can not be identified as a tolerogenic tree the only standard cell projections.Part two. Construct p65 low expression lentivirus vector and detect biological characteristic of dendritic cells transfected by lentivirusObjectiveDesigned for p65 low expression of siRNA sequences, construct lentivirus vector system and verify their silencing effect on the target gene, induce differentiation of dendritic cell by lentivirus transfected into tolerogenic dendritic cells, and research the biological properties of the transfected dendritic cells.MethodsAccording to siRNA design principles and methods, design three specifically interfere with p65 gene sequence of rat and a negative control sequence; transfected 293T cells in vitro; base on reverse transcription polymerase chain reaction test and Western blotting assay, analyze the protein expression and mRNA expression levels of p65; after lentivirus transfected or/and LPS stimulation, dendritic cell phenotype was discussed by flow cytometry; base on mixed lymphocyte reaction experiments, analyze the biological characteristics of tolerogenic dendritic cells; base on reverse transcription polymerase chain reaction test and ELISA assay, mRNA expression and protein expression levels of IL-10, IL-17 and IFN-y was analyzed; verify direct impact of p65 by chromatin immunoprecipitation and luciferase reporter.ResultsDesign and Synthesis of siRNA targeting sequence p65 low expression can effectively inhibit p65 and NF-κB protein expression levels of cell, significantly reduced mRNA expression levels of p65 (p<0.05); protein blot results indicate p65 and NF-κB band was stronger control than the lentivirus group; mixed lymphocyte reaction experiments LPS stimulation (LPS) compared to the other three groups of rat allogeneic T cell proliferation was significantly stimulatory effect (p<0.05); streaming cytometry analysis of LPS stimulation (LPS) in the detection of CD80, CD86 and CDllc+MHC-ii were significantly higher than in the (p<0.05) in the control group (control), lentivirus group (Lv) and lentivirus after transfection LPS stimulation group (Lv+LPS); cell supernatant p65, IL-17 and IFN-y mRNA and protein expression of test results in LPS stimulation group (LPS) was significantly higher (p<0.005) the other three group; mRNA and protein expression of test results in the control group (control) cell supernatant of IL-10 protein expression was significantly higher (p<0.005) LPS stimulation (LPS), lentivirus group (Lv) and lentivirus transfection after LPS stimulation group (Lv+LPS), LPS stimulation (LPS) was significantly higher (p <0.005) lentivirus group (Lv) and lentivirus after transfection, LPS stimulation group (Lv+LPS); fluorescein luciferase reporter gene results in IFN-y, IL-17 and lentivirus group RLU index IL-10 were significantly lower than the control group; chromatin immunoprecipitation experiments in lentivirus group of IFN-y, IL-17 and IL-10 strip relative input lentivirus group and the control group were significantly weakened.ConclusionLentiviral vector can effectively reduce p65 and NF-κB protein expression levels and significantly reduced mRNA expression levels of p65. Whether or not LPS stimulation, transfected bone marrow-derived dendritic cells can maintain immature dendritic cells phenotype and biological characteristics of tolerogenic dendritic cells. Lentivirus transfected bone marrow-derived dendritic cells reduce protein expression and mRNA expression levels of IFN-γ and IL-17. p65 can directly bind to the IL-10, IL-17 and IFN-y promoter region and initiate transcription and translation expression of IL-10, IL 17 and IFN-γ.Part three. The effect and mechanism of p65 low expression induced tolerogenic dendritic cell in allograft keratoplasty immune rejectionObjectiveDiscuss the mechanism and influence of lentivirus transfected bone marrow derived dendritic cells induce immune tolerance in allogeneic orthotopic penetrating corneal allograft.MethodsEstablish orthotopic keratoplasty model of allograft rat, donor and recipient rats were randomly divided into four experimental groups, respectively inject bone marrow derived dendritic cells or transfected lentivirus dendritic cells in preoperation or/and postoperation, observe graft survival, corneal neovascularization and corneal opacity and establish the evaluation system; base on immunohistochemical staining, analyze expression level of related cytokines expression; base on flow cytometry, analyze expression level of regulatory T cells; base on reverse transcription polymerase chain reaction test and ELISA assay, analyze mRNA expression and protein expression levels of IL-10, IL-17 and IFN-γ.ResultsThe graft mean survival time of control group was 20 ± 6.55 days BMDC group was significantly longer than the control group (13.47± 2.56 days), the average survival time of Lv-p65-DC group and Lv-p65-DC-cont group are more than 30 days; from the 12th day, the opacity of BMDC group was significantly higher than the other three groups (p<0.05), in the third week after surgery, the opacity of Lv-p65-DC group were lower or higher than the control group and Lv-p65-DC-cont group; the first 10 days, angiogenesis scores of Lv-p65-DC-cont group were significantly lower than the other three groups; immunohistochemistry results showed that the expression of IL-17 is specific and significantly enriched in the corneal neovascularization wall; regulatory T cells expression level of BMDC group and control group were higher than the other two groups; p65 mRNA and protein expression levels of Lv-p65-DC group was significantly higher than Lv-p65-DC-cont group in the third week; IL-30 mRNA and protein expression levels of the control group was significantly higher than the other three groups in the first week, IL-10 mRNA and protein expression levels of the control group and Lv-p65-DC group was significantly higher than Lv-p65-DC-cont group in the third week; mRNA and protein expression characteristics of IL-17 and IFN-y is more approximate, Lv-p65-DC group and Lv-p65-DC-cont group were significantly lower than BMDC group and control groups in the first week; mRNA and protein expression levels in the control group and Lv-p65-DC group were significantly higher than Lv-p65-DC-cont group in the third week.ConclusionWith inflammatory signals stimulated, non-transfected bone marrow derived dendritic cells gradually differentiated into mature dendritic cells in vivo. The process promote immune rejection. Before operation, injection of transfected dendritic cells can induce regulatory T cell activation, prolong graft survival and reduce corneal angiogenesis and the opacity grafts. Lv-shRNA-p65 can reduce the expression level of IL-10, IL-17 and IFN-γ. This lentivirus injection method has time-dependent. |
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