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Research of Targeting Cytotoxity Platform Based on T Epitope and Dentritic Cell-oriented

Author: LuoJin
Tutor: YanXiaoJun
School: Fourth Military Medical University
Course: Biochemistry and Molecular Biology
Keywords: Dendritic cells Epitope peptide Antigen-presenting Cytotoxic T cells MHC-Ⅰ pathway Immune response
CLC: R392
Type: PhD thesis
Year: 2008
Downloads: 259
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Abstract

Continuous progress and breakthroughs with the theory of immune recognition, knowledge of molecular biology and electron microscopy techniques, a large number of studies have confirmed that the T cell antigen recognition combined with MHC molecules presenting antigen peptides. Effective antigen epitope peptides by dendritic cells presenting MHC-Ⅰ / MHC-Ⅱ pathway caused by the body's immune response. In recent years scientists have suggested that composite epitope or reconstructive epitopes can enhance the function of the single epitope. With the DC vaccine clinical application of further in-depth research to find effective T cell epitopes to enhance DC vaccine by more and more attention to the role of the anti-tumor and treatment of infectious diseases. How to make a foreign antigen by MHC-Ⅰ way to induce CD8 CTL response DC key clinical applications. The topics designed mainly for the cause of the viral disease, intends to select the hepatitis B virus-derived hepatitis disease models, cellular immune attack of hepatitis B virus core protein as a target protein, the through software prediction method analysis the biological characteristics of the protein and the T-cell epitope peptide, epitope modified obtained by the design and synthesis. DC culture create a standardized platform; lay the foundation for the clinical application through experiments explore the epitope peptide of DC function and its mechanism for DC. The experiment is divided into four parts, as follows: the first part of the design of the T-cell epitope peptide screening purposes forecasts HBV core protein of the general biological characteristics of the protein fragment HLA-2 restricted CTL epitopes. Choose from Genebank type B (adw) HBV core protein of HBV core antigen with the software the CLC Protein Workbench3 version SignalP3.0 software (http://www.cbs.dtu.dk/services/SignalP-2.0/) General biological characteristics; encoding proteins predicted using remote SYFPEITHI prediction database PREDEPP database motif and the polynomial method common protein encoded by the HBV core antigen HLA-A2 restricted CTL epitope prediction. HBV core protein results by software analysis, clear the transmembrane region, the signal peptide of the protein, the subcellular localization, antigenic sites. Filter the five T-cell epitope peptide. Conclusion prediction scheme used in combination can improve the accuracy of the T-cell epitope peptide screening. Second portion identification of T-cell epitope peptide, modified with synthetic purposes identified screened five peptide biological characteristics, modified and synthesized with the antigen-binding properties of the best peptides for further study of exogenous epitope peptide into small molecules cells stimulate the MHC1 class reaction to lay the foundation. Method with the performance of the antigen binding experiment separately from the affinity binding stability and MHC-peptide complex stability 5 Identification of the epitope peptide and the antigen-binding properties, screening out the binding properties of the best of the two peptide, and helper T -cell epitopes, B cell epitopes and palmitoyl-modified, Fmoc program synthesis. The results are screened out two T-cell epitope peptide, synthesis of 9 peptide, constituting the 11 groups of the epitope peptide. Conclusion antigen binding performance experiments and the Fmoc synthesis program can be completed Identification and synthesis of the antigen peptide. The third part of the human peripheral blood monocyte-derived dendritic cells into the platform structures to create a standardized, scale of operation of DC culture, transformation and identification platform. Methods of human peripheral blood PBMC with CD14 beads marker, in the magnetic field separated CD14 PBMC; sorting the cells obtained in the DC culture bag by adding IL-4, GM-CSF co-culture, 10 days, collect cell count the, Typanlan staining activity, flow cytometry cell phenotype, scanning electron microscopy cell morphology. Results under an optical microscope, the of dendritic cell suspension growth, ranging in size, very irregular shape, extending around the different number of varying thickness, very different patterns of cytoplasmic processes. CD14 PBMC in the initial blood in the ratio of 14%, 94.8% and after separation of the concentration of the purified CD14 cells. In cell suspensions were cultured for 10 days, since the majority of the cells is converted to DC, the molecular surface marker changes of CD14-cells in a concentration of only 0.65%; DC cell surface in the culture out of the high expression of HLA-I (70.43%) , HLA-Dr (0.65%), CD80 (89.45%), CD86 (86.32%), part of the expression of CD40 (29.61%). These cell surface markers are human PBMC into DC characteristic molecular. Scanning electron microscopy showed the dendritic cells are fixed remains the projection of its characteristic, so that the cells were stellate, polygonal, or highly irregular shape. Projections of varying lengths, branch formation level 1-4 sub protrusions. Conclusions of CD14 cells isolated and purified from PBMC by the addition of cytokines in the cell culture bag non-adherent type culture of high purity DC. The fourth part of DC-mediated targeted killing function and mechanism explore Objective To compare the biological function of the DC load a different epitope peptide screened killing function epitope peptide combination, from the molecular level of micro combinatorial peptide The functional differences. Method with four thiophene azole blue law experimental detection of cytotoxic function; ELISPOT experimental detection load different table bit of DC to stimulate T cells secrete γ-of IFN's function; atomic force microscopy analysis of DC after small molecule peptide role of surface fine structure change; confocal laser scanning microscope The analysis of small peptides into the cells after different time performance; the immunofluorescence microscopy DC intake peptide process. Results cells were T1, T1 T2, T1 Th, T1-AAA-Th, pal-KSST1, pal-KSST1AAATh, T1 B, pal-KSST1Th, T1 Th, group B short peptide treatment, cell activity than the control group had a significantly elevation (p lt; 0.05), which the the T1 B group peptide showed a very significant increase (p lt; 0.01) cell activity can be after T2, Th, T1Th peptide treatment, cell activity than the control group significantly reduced (p lt; 0.05), T2 group peptide showed a very significant reduction in cell activity. Filter out the epitope peptide T1 can be raised DC on T cell stimulating ability (P lt; 0.01), while after palmitoyl serine modified epitope or B cells, Th is synergy of the T-cell epitope load DC stimulation capability also have the same performance ( P lt; 0.01), regardless of helper T cell epitope or B-cell epitopes for T1 are synergistic. The helper T cell epitope directly coupled to the T1 epitope, destroyed the T1's ability to stimulate DC. Even in the T1 epitope and Th internally by the AAA connection, and serine palmitoyl modification will not exhibit obvious function than individual T1 epitope enhancement. The results show that small peptides can in 30 minutes DC rapid uptake, immunofluorescence microscopy revealed that peptide uptake is completed within 5min; pass to the dendritic uptake of small peptides by the completion of the cell body and dendrites of confocal laser The peptides concentration of relatively low cell body. Within 12h after the small molecule peptide cellular uptake, and never made into the nucleus. Load peptides under a transmission electron microscope, DC mitochondrial increases, increased ribosomes and endoplasmic reticulum. And MTT activity detection experiments show that these peptides function of DC activation increases. Conclusion DC after different peptide cell activity will produce some degree of change. Mainly due to increased killing activity of short peptides to T lymphocytes through DC activation of cellular immune pathway play CTL effect. Palmitoyl modified can upregulate the function of the T-cell epitope; B cell epitope and Th can be enhanced through epitope synergies among the effect of the T-cell epitope peptide activated CTL. AFM surface morphology changes observed in cells affected by exogenous small molecule peptide primary structure, but also directly affect the activity of the cell. Small peptides as the genetic material involved in the transmission of information of the cell, this phenomenon epitope peptide as a vaccine, co-cultured with DC reinfusion after the body of the security to provide a theoretical basis. Software prediction is a viable T-cell epitope peptide screening programs, greatly reduce the workload of the experiment, the epitope peptide of the different categories of the synergy between the biological function for the up-regulation of the DC is greater than the epitope between The coupling effect. Palmitoylation epitope peptide can also be raised to DC for T-cell stimulatory capacity. Different small molecule peptide is not the same, the influence on the fine structure of the DC surface the generated CTL killing effect is also different.

Research of Targeting Cytotoxity Platform Based on T Epitope and Dentritic Cell-oriented

Abstract

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