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The Emerging Role of Tim3 and Tim4 in Immunoregulation in an Experimental Mouse Heart Transplant Model

Author: FangZeMin
Tutor: ChenZhongHua
School: Huazhong University of Science and Technology
Course: Surgery
Keywords: Graft Collagenase Ⅱ Transplant heart infiltrating lymphocytes Flow cytometry Tim3 Acute rejection T lymphocytes Galectin-9 Heart transplant Mice Tim4 Tim1 Tolerance
CLC: R392
Type: PhD thesis
Year: 2010
Downloads: 115
Quote: 0
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Objective To establish a simple and efficient separation of mouse heart graft infiltrating lymphocytes infiltrating lymphocyte subtypes heart transplant, the use of flow cytometry. The established mouse cervical heart transplantation model, the the points experimental group (allograft group: Balb / c mice were used as donor, C57BL / 6 mouse receptor) and the control group (isograft group: donor and recipient C57BL / 6 mice). 3 days after transplantation, 5 and 7 days of access to transplant heart rejection of the case, the part of the graft biopsy observation, the remaining graft Shredded and improved type II collagenase digestion (250 U / ml, 30 -40 min) digested heart, and mononuclear cells were isolated by Ficoll density gradient centrifugation (800g × 20 min), based on calcium ion analysis of fluorescent dye Indo-1 cell activity, and finally to the fluorescence-labeled antibody of CD4, CD8, CD44 and CD62L, Foxp3 flow cytometry staining, flow cytometry graft-infiltrating lymphocyte subsets. Result, the separation of the number of mononuclear cells in the graft stable at 1 × 106 after 7 days reached a peak in the number of mononuclear cells separated from the number and severity of rejection related. The lymphocytes accounted mononuclear cells isolated from the ratio of (31.9 ± 2.3)%, (95.1 ± 2.1)% activity. The majority of infiltrating T cells within the graft to effector T cells, CD4 / CD8 ratio gradually reduced with the advance of the rejection. Conclusion The method is single transplanted heart was digested with collagenase, using Ficoll density gradient centrifugation method, reducing the damage cardiac graft infiltrating lymphocytes, cells obtained can be further used for flow cytometric analysis of their phenotype for directly detecting infiltration into lymphocyte subtypes within the graft to provide an efficient and reliable method, is a worthy means of immunological studies. The purpose of detection after transplantation Tim3 mRNA the graft expression changes and Tim3 in different parts in the body of the recipient T lymphocytes investigate the expression and its relationship to acute rejection. Established mouse heart transplantation model, divided into experimental group (allograft group: Balb / c mice were used as donors and C57BL / 6 mouse receptor) and the control group (isograft group: donors and recipients are C57BL / 6 mice). 3 days after transplantation, 5 days, 7 days and 9 days for graft, using real-time quantitative RT-PCR analysis Tim3 mRNA expression changes in the graft. After 3 days and 6 days separated by the peripheral blood, spleen, draining lymph nodes, lymphocytes in the graft, Tim3 positive cells in the proportion of CD4 and CD8 T cells by flow cytometry. The results with the genome the grafts Tim3 expression is very low. The allogeneic group transplanted graft within 3 days after Tim3mRNA expression was significantly higher, and Tim3 mRNA expression with the rejection of the conduct of significantly elevated (P lt; 0.01), reached a peak before the graft completely excluded, The subsequent expression gradually reduce. The peripheral blood and spleen of transplant recipients Tim3 positive cell ratio was no significant change (P gt; 0.05), draining lymph nodes Tim3 / CD4 ratio increased slightly (P <0.05), graft Tim3 / CD4 and Tim3 / CD8 ratio was significantly higher (P lt; 0.01). Transplant after 3 days and 6 days draining lymph nodes Tim3 / CD4 ratio of the difference was not statistically significant (P gt; 0.05), and six days after graft Tim3 / CD4 Tim3 / CD8 ratio significantly higher than 3 days (P lt; 0.01). Conclusion graft Tim3 mRNA expression in mice completely different genetic heart transplant rejection progress dynamic. Group recipients of allogeneic graft-draining lymph nodes and graft Tim3 cell ratio increased graft increased more significantly. Objective To observe Galectin-9 allogeneic heart graft survival time to explore Galectin-9 activation immunomodulatory effects Tim3-Tim3L pathway in mouse heart transplantation model. Established a mouse cardiac allograft model, divided into experimental group and control group. Experimental group recipients after seven days in a row to give stable Galectin-9 protein in the control group were given PBS. Observation of cardiac allograft survival time. After 7 days for the experimental group and a control group of cardiac allograft biopsy observed rejection, graft-infiltrating CD4 and CD8 T cell numbers observed by immunohistochemistry. Real-time quantitative RT-PCR to detect graft Tim3, IFN-γ and IL-17mRNA expression. Flow cytometry draining lymph nodes and graft Tim3 proportion of cells and peripheral blood Th1 and Th17 cell ratio. Results Galectin-9 protein treated cardiac allograft survival time was 22.7 days in the control group, only 7.2 days, Galectin-9 treatment group graft survival time was significantly longer (P lt; 0.05). The biopsy revealed Galectin-9 treatment group graft rejection significantly reduced infiltration of CD4 and CD8 T cells was significantly reduced. Galectin-9 treatment of graft Tim3, IFN-γ and IL-17mRNA decreased expression. Draining lymph nodes and graft Tim3 cells reduce the proportion of peripheral blood Th1 and Th17 cell ratio is also reduced. The the conclusion short term Galectin-9 treatment can significantly prolong allograft heart graft survival time, and the mechanism is to reduce graft lymphocytic infiltration and Tim3 programmed cell death is triggered reduce recipients vivo Th1 and Th17 cell responses. Objective To observe the blocking Tim4 mouse heart graft survival time, to explore Tim4 role in the the pathway induced Tim4-Timl immune tolerance. Established a mouse cardiac allograft model, to give the the recipient anti Tim4 antibody to observe its impact on graft survival time, in vitro mixed lymphocyte culture detection the anti Tim4 antibody alloreactive T-cell proliferation role. The Compare Tim4 a genetic defect and wild-type heart graft survival time of the receptor in vivo, biopsy judgment of exclusion, immunofluorescence staining of graft-infiltrating lymphocytes. Real-time quantitative RT-PCR was used to detect the expression of immune-related genes in the graft, the flow cytometry graft infiltrating lymphocyte subtypes changes. Adoptive transfusion recipients in vivo effector T cells and regulatory T cells to Babl / c SCID mice to observe its impact on skin graft survival time. Given a small dose of rapamycin to observe the impact of the genetic defect on Tim4 graft survival time, and cleared by preoperative administration of anti-CD25 antibodies recipient body Treg clear the role of Treg in the experiment. Results the anti Tim4 antibody can inhibit the mixed lymphocyte culture system alloreactive T cell proliferation, prolong graft survival time. The donor Tim4 genetic defect significantly reduced rejection, reducing graft effector T cell infiltration, increase the number of regulatory T cells, thereby significantly prolong graft survival time (P <0.01). The Tim4 genetic defects can reduce the activation of effector T cells and increase the number of Treg, but had no significant effect on effector T cells and Treg function. Combined with low dose the rapamycin induced Tim4 gene defects long-term graft survival. Tim4 genetic defect or / and a small dose of rapamycin to prolong graft survival time dependence of Treg exist. The conclusion Tim4 allogeneic immune response play an important immunomodulatory role, blocking Tim4-Timl pathway can significantly prolong heart graft survival time.

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