| ObjectivesTo establish the methods of isolation, purification and propagation of F344 rat bone marrow derived dendritic cells (BM-DC), study the morphological structure and bio-immunological characteristics of BM-DC during the maturation and differentiation and investigate the immunological tolerogenity of BM-DC propagated with TGFβ1. To reconstruct the mammalian plasmid TGFβ1-pcDNA3 and ASTGFβ1-pcDNA3 and tranfect them into BM-DC with lipofectin DOTAP, study the effects of TGFβ1 gene transfection on the differentiation, maturation and immunological functions of DC. To observe the migration and homing of the transfected DCs into the T lymphocyte area of spleens and lymph nodes a week after injection into the allogenic Lewis rat recipients and detect the apoptosis and expression of hTERT of T lymphocytes. To investigate the influence of TGFβ1-DC injection on the grades of cardiac transplantation rejection and the mean survival time of cardiac allografts, evaluate the effects of TGFβ1-DC on the expression of several chemokines related such as RANTES (regulated by activation, normal T cell expressed and secreted ) , MCP-1 (monocyte chemokine protein-1), MlP-lα (macrophage inflammatory protein-1α), FKN (Fractalkine) and its receptor CX3CRl, etc.MethodsDC progenitors were isolated from bone marrow of F344 rats, and propagated in culture medium with GM-CSF and TGFβ1 or IL-4. The immunological characteristics of DC were assayed with FACS, MLR and immunocytochemistry. After 9 days' culture, the TGF-β1 cultured cells were collected for TGF-P1 gene transfection. After one day's culture, the cells were injected (2xl06, v.v.) into the allogenic Lewis rat, which is going to be the recipient of heart transplantation 7 days later. The apoptosis of T cells in recipient spleen was analyzed by TUNEL staining. The animals weredivided into five groups: Group A (isogenic transplantation), group B (allogenic transplantation), group C (TGFβ1 DC injected), group D (ASTGFβ1-DC injected)) and Group E (EL-4 DC injected). The cardiac allograft survival was determined directly by daily palpation of the cardiac impulse through the abdominal wall, and rejection was considered as cessation of a palpable heart beat. Acute cardiac rejection grades were valued by the Stanford standard of ISHLT (1990). Immunohistochemistry was performed to analyze the expression of the related chemokines RANTES, MCP-1, MIP-1α Fractalkine, and its receptor CX3CR1 in cardiac allografts.Results1. Rat bone marrow mononeuclear cells grew with rrGM-CSF alone or rrGM-CSF+IL-4. Developing DCs were subsequently analyzed for the molecular phenotypes after 6 or 10 days' culture. With GM-CSF alone, the proliferation rate of DCs was low and FACS staining showed that only a few cells were identified to express weak of MHC class II (RT1B) and costimulatory molecules. However, the addition of rrIL-4 to the culture medium strongly induced the formation of growing cell aggregates, which were essential for the generation of rat BM-DC. Cell aggregates slightly adhered to the bottom surface of the plates by day 5-6 and continuously increased in size and number. At day 8-9, the cell aggregates were more loosely adherent and could easily be dislodged with a pipette. After 24 hours, large numbers of BM-DC with their typical morphology could be identified in the supernatant. At an early stage of development (day 6), BM-DC showed an intermediate level of RT1B and low levels of the costimulatory molecule B7-2. During the period of further culture, the expressions of RT1B, costimulatory molecule B7-2, integrin OX62, and intracellular adhesion molecules (ICAM-1) were observed continuously increased.2. It was found that the DCs cultured with the supplement of TGFβ1 in the medium significantly decreased their surface expressions of RT1B and costimulatorymolecule B7-2 and slightly inhibited the levels of ICAM-1 and integrin OX62. Besides, the yield of cells was lower than that propagated with rat IL-4. The capacity of sti... |
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