Dissertation > Medicine, health > Basic Medical > Medical Immunology > Transplantation immunology

Investigation of T Cellular Immunologic Tolerance Induced by mIDO Gene-transfected Tissue Engineering Chondrocytes

Author DuanXiaoHong
Tutor CuiPengCheng;JiangXun
School Fourth Military Medical University
Course Department of Otolaryngology,
Keywords Tissue Engineering Chondrocytes Indoleamine 2,3 - dioxygenase Transplantation Immunology Immune tolerance
CLC R392.4
Type Master's thesis
Year 2008
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The repair of cartilage defects is a major problem in clinical, due to limited sources of autologous chondrocyte transplantation of tissue engineered cartilage transplant will be an important way to solve cartilage repair the damage, however, the tissue-engineered cartilage with immunogenicity. The issues contemplated by transfection of indoleamine 2,3 - dioxygenase (indoleamine 2,3-dioxygenase, IDO) gene in order to change the purpose of local T cell immune response to negatively regulate chondrocyte transplantation, induction of transplant recipients T cell immune tolerance to allogeneic tissue engineered cartilage graft, prevention of autoimmune clinical allograft rejection technology for the development of transgenic lay the foundation for the realization of tissue engineering seed cells can be used in different immune genetic background patients overcome of MHC (major histocompatibility complex, major histocompatibility complex) polymorphism and multiple genes are formed allogeneic immune barrier to reach the seed cells of tissue engineering \a partial solution to immune rejection from graft new. The main content and the results are as follows: (a) mice were isolated and cultured chondrocytes 1.C57 mouse articular cartilage Get: 37 ℃ in vitro by 0.2% Ⅱ collagenase digestion 3 ~ 4 h after centrifugation, the collection can get for the experiment with a large number of cartilage cells, cartilage cell viability trypan blue staining digestion method to obtain more than 95%. 2 primary cultured cartilage cells, MTT assay cartilage cell growth curve, found the first 2 to 5 days after the adherent chondrocytes into the exponential growth phase, the bottom of the bottle can be covered with about 7 d, but with the passage number of cells increase significantly longer generation time of cells to form. 3 cartilage cells secreted extracellular matrix, toluidine blue staining showed blue metachromasia reaction to obtain the cartilage cells of the present experiment after toluidine blue staining, and found that the cell purity above 85%, but with an increase in the number of passages The decline in cartilage cells secrete extracellular matrix, to select 2,3 generation cartilage cells are used in the best period of the experiment. (B) the chondrocytes pEGFP-N1-IDO plasmid transfection and gene expression. Optimized liposome and plasmid ratio (2:1), plasmid pEGFP-N1-IDO liposomal transfection to primary the chondrocytes cultured chondrocytes after transfection still adherent growth. Fluorescence microscopy and laser scanning confocal microscopy showed that the monitoring of EGFP expression in chondrocytes after transfection, the green fluorescence is evenly distributed throughout the cell, and 12 h after transfection, and strong expression at 48 h. EGFP was expressed in the cytoplasm and nucleus, but nuclear expression was significantly higher than in the cytoplasm. 3 at different time points after transfection collection of cartilage cells, paraformaldehyde, flow cytometry EGFP expression in chondrocytes, with time after transfection, the transfection efficiency increased first and then decreased trend. 24 h after transfection, peaked at 48 h after transfection efficiency can be as high as 36%. Chondrocyte gene 48 h after transfection, RT-PCR RNA levels can be detected to mIDO expression, compared with the optical density of β-actin, the relative expression levels of the gene in chondrocytes stable. (C) mIDO transfected chondrocytes in vitro proliferation of T lymphocytes. Using high pressure liquid chromatography (HPLC) analysis of the content of tryptophan in the culture medium of chondrocytes mIDO transfection chondrocytes alone group, 24 h after color The acid content of 4.93 ~ 5.71 mg / L, an average of 5.3 ± 0.39 mg / L, and gene transfection chondrocytes group does not detect tryptophan. Tryptophan degradation in the cell culture fluid, and also along with the increase in kynurenine metabolites, the cell supernatants tryptophan eliminate Kinetic studies indicate that the transfected gene cartilage cell supernatants tryptophan the degradation significantly faster in chondrocytes alone group. 2 transgenic chondrocytes in vitro mixed lymphocyte reaction to 2 × 105 3 000 radγ ray irradiated spleen cells as antigen presenting cells and 3 × 105 lymph node lymphocytes as the immune response cells, after the mixed reaction # 5 d, 6 d, 7 d, 8 d by MTT assay groups lymphocyte proliferation. The results show that the lymphocytes than that spleen cells group, transgenic and non-transfected genomic lymphocyte proliferation (P lt; 0.02), and turn the IDO genome lymphocyte proliferation compared with non-transgenic group slowed down (P lt; 0.05). Mixed lymphocyte reaction, the immune response of CFSE-labeled cells co-cultured proliferation response of T cells by flow cytometry after 96 h, transgenic group and non-transfected genomic response cell proliferation (P lt; 0.01) 9 cells were formed. IDO gene 96 h after transfection group overall proliferation index was 1.122 ± 0.017, simple lymphocytes negative control group was 1.201 ± 0.026 1.026 ± 0.016, positive simple chondrocytes group than the positive control group, IDO genome allogeneic T lymphocytes proliferation significantly inhibited. Conclusion: digestion method available for a large number of experiments with primary cultured chondrocytes, and 3rd generation cartilage cells are used in the best period of the experiment; liposome is expected to become the primary cultured chondrocytes gene transduction IDO stably expressing cells, and is expressed mainly in the cytoplasm; transgenic cells cultured in vitro, significant degradation of tryptophan concentration in the culture medium, as carrier, mIDO import primary cultured chondrocytes of EGFP monitoring IDO and inhibit the degradation of tryptophan in the local microenvironment concentration to provide a scientific basis allogeneic T cell proliferation; express transfected chondrocytes mIDO significantly inhibited allogeneic T cell proliferation in vitro mixed lymphocyte reaction gene transfection chondrocytes to inhibit the proliferation of T cells through the degradation of the concentration of tryptophan in the local microenvironment provides a guideline to prolong graft survival time.

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