The Experiment Study On Immunity-tolerance Of The Transplanted Heart Induced By Immature Dendritic Cell Transfected With TGF-β Gene

Part 1. The construction of Heart Transplantation Modelof mouse to cervical and comparison of the two techniquesObjectives: Use two techniques to construction the model of heterotopic heart transplantation of mouse to cervical, to establish the foundation for further study and to comparison the advantages and disadvantages of the two techniques.Methods: The experiment was devided into two groups, allogennial graft group(n=39) and isograft group(n=25), the allogennial graft heart transplantation was operated among Kunming monse, the isograft heart transplantation was operated among BALB/c mouse. We operated 39+25 times. Dragging the segmental eqidural catheter to make it into vessel cannula and used during the operation. The operation was performed by a single surgeon with an operation microscope with 10 times magnification. The donor and the recipient were operation anternativelly;at first ,we make the artery cannula and venous cannula,then deal with the donor heart,at last, we use sewing-technique and cuff-technique,anastomose the common carotis cannula of the recipient to the ascengding artery of the donor heart and the external jugular vein cannula to the pulmonary cannula respectively.Results: We operated 39+25 times. All the transplatated heart recover beat again. The survival rate 3 days after operation was about 72% and 92%. The ischemic time of the donor heart was less 30 minutes.The average survival period of the allogennial graft heart was 8.3±1.3 days.Pathlogy examination show that happened typical rejected reaction. The isograft heart can survive long. Pathlogy examination showed no rejection reaction happened.Conclusion: By cuff-technique, we shorten the ischemic time, simplify the operation. Success ratio of the operation is higher and the vessel unobstructing ratio after the operation is better. The complication ratio isn't increased and the total operation time isn't prolonged conspicuously. It is a preferred way to construct the model for new recruiter.Part 2. The generation of Immature Mouse Myeloid Dendritic Cells and identification their bionomics charactersObjectives: This study was performed to establish a simple method to culture immature dendritic cells derived from mouse bone marrow cells and observe their biological features.Methods: Mouse DCs were generated with high doses (200U/ml) or low doses (50U/ml) from bone marrow cells separately, the phenotype and functional properties of these DCs were compared through fluorescence-activated cell sorting (FACS) analysis and mixed lymphocyte reaction (MLR), and expression levels of IL-10 mRNA and TGF-β mRNA in culture cells were detected using semi-quantitive RT-PCR.Results: DCs cultured with high doses were a mixture of mature DCs and immature DCs, they had a mature phetotype:CD11c⁺, CD25^±, CD80⁺, CD86⁺, MHCII hi, and can stimulate allogeneic T cell responses strongly, whereas DCs generated with low doses were phetotypically immature DCs (CD11c⁺, CD25^-, CD80^- CD86^-, MHC II^low), and can't stimulate allogeneic T cell response efffectively. The expression level of IL-10 mRNA in immature DCs was much higher than the other cells, no significant difference was found in the expression levels of TGF-β mRNA in culture cells.Conclusion: Low doses of GM-CSF can stimulate immature DC, this kind of DC cell can't stimulate allogeneic T cell responses effectively. They express IL-10 mRNA at high lever, perhaps because they can excrete IL-10 and this is maybe the cause which induced immunity tolerance.Part 3. The construction and identification of TGF-β gene eukaryotic expression vector of mouse Objectives: The construction and identification of eukaryotic expression vector of TGF-β gene of mouse.Methods: Drow off the bone marrow of donor BALB/c mouse, extract the mRNA of the total cell.Amplification the TGF-β structure gene sequence by RT-PCR technique. Ligation TGF-β structure gene fragment to plasmid pcDNA3.1, to construction the recombinant plasmid pcDNA3.1- TGF-β with gene engineering technique. Then transfect the recombinant plasmid pcDNA3.1- TGF-β into the Escherichia coli by hypothermy calcium-chloride technique. Choose the TGF-β gene positive-expressed Escherichia coli to extract recombinant plasmid to identify by rectriction enzyme reaction and sequence analysis.Results: From the recombinant plasmid pcDNA3.1- TGF-β, we obtained the DNA fragment according with the expectation product by RT-PCR technique. And the DNA fragment analysis is the same to Genebank.Conclusion: Constructed the recombinant plasmid pcDNA3.1- TGF-β succeedly, it contains all the structure gene of TGF-β of mouse.Part 4. The study on immunity-tolerance of the transplanted heart induced by the immature DC transfected with TGF-β geneAbstract:Objectives: Observe the influence of immature DC transfected with TGF- β gene on prolonging the survive period of allogenial graft heartMethods:Using low of GM-CSF as stimulator, we culture the immature DC derived from bone morrow of mouse, then transfectd recombinand plamid TGF-P-pcDNA3.1 into the cell by lipidosome.So we obetain the immature DC transfected with TGF-p-pcDNA3.1.The experiment was designed as follow:it has two groups:transfected DC group vs single DC group. â‘ Dectect the TGF- β mRNA of DC of the two groups by RT-PCR technique. â‘¡Dectect their ability to induce allogeneic T cell responsiveness by mixed lymphocyte reaction (MLR).â‘¢A week before operation, we infuse two kinds of DC to the recipients, then observe the influence of the cells on prolonging the survive period .After 7days of the operation, transplanted heart were reciped for pathology examination.Results: The DC transfected with recombinand plamid can express TGF- β mRNA at high lever than the single DC. The transfected DC cann't stimulate T cell responsiveness in vitro, wherever the single DC sitmulate T cell responsiveness at very low lever .As to the influence on the survive period of the transplanted heart,. the transfected DC can prolong the time than single DC group (22.3±3.3d vs 15±2.2d), two of them are longer than control group(7.1±0.8d).Pathology examination shows that: rejection reaction was imponderable of the transfected DC group (Stanford 0^l level) .rejection reaction was slightly of the single DC group (Stanford 1² level), rejection reaction was severly of the control group (Stanford 3⁴ level).Conclusion: DC cultured with low dosed of GM-CSF only express TGF- β mRNA at low level, can stimulate T cell responsiveness at low level.After transfected with TGF-β, DC can express TGF- β mRNA at higher level. It stimulate T cell nonresponsiveness. Infuse the DC transfected with TGF-β gene cultured with low dosed of GM-CSF a week before operation, can prolong the survive period of the transplanted heart. It stimulates T cell nonresponsiveness. Perhaps it is the important reason that induced Th1/Th2 immunity deviation.