Tim3和Tim4在小鼠心脏移植模型中的免疫调节作用机制研究

发布时间：2018-04-03 15:25

本文选题：移植物　切入点：胶原酶Ⅱ　出处：《华中科技大学》2010年博士论文

【摘要】： 目的建立简便而高效的分离小鼠心脏移植物内浸润淋巴细胞的方法,使用流式细胞仪检测移植心内浸润淋巴细胞的亚型。方法建立小鼠颈部心脏移植模型,分实验组(同种异体移植组：Balb/c小鼠为供体,C57BL/6小鼠为受体)和对照组(同系移植组：供受体均为C57BL/6小鼠)。移植术后3天,5天和7天获取移植心,将其中一部分移植物进行病理切片观察排斥反应情况,剩余移植物剪碎并采用改进的Ⅱ型胶原酶消化法(250 U/ml,30-40 min)消化心脏,然后用Ficoll密度梯度离心法(800g×20 min)分离单个核细胞,以钙离子荧光染料Indo-1分析细胞活性,最后以荧光标记抗体CD4, CD8, CD44, CD62L, Foxp3进行流式细胞染色,流式细胞仪检测移植物内浸润淋巴细胞亚群组成。结果移植物内分离的单个核细胞数量稳定在1×106以上,术后第7天数量达到最高峰,分离所得的单个核细胞数量与排斥反应严重程度相关。淋巴细胞占分离所得单个核细胞的比例为(31.9±2.3)%,活性为(95.1±2.1)%。移植物内浸润的T细胞大部分为效应性T细胞,CD4+/CD8+比值随着排斥反应的进行逐渐降低。结论本法单用胶原酶消化移植心,采用Ficoll密度梯度离心法,减少了对心脏移植物内浸润淋巴细胞的损伤,获得的细胞可进一步用于流式分析其表型,为直接检测浸润到移植物内的淋巴细胞亚型提供一种高效和可靠的方法,是一种值得推广的免疫学研究手段。目的检测Tim3 mRNA在移植后移植物内的表达变化及Tim3在受者体内不同部位T淋巴细胞上表达,探讨其与急性排斥反应的关系。方法建立小鼠心脏移植模型,分实验组(同种异体移植组：Balb/c小鼠为供体,C57BL/6小鼠为受体)和对照组(同系移植组：供受体均为C57BL/6小鼠)。移植术后3天,5天,7天和9天获取移植物,采用实时定量RT-PCR检测Tim3 mRNA在移植物内的表达变化。术后3天和6天分离受者外周血、脾脏、引流淋巴结、移植物内淋巴细胞,流式细胞仪检测Tim3阳性细胞在CD4+和CD8+T细胞中的比例。结果同基因组移植物内Tim3表达很低。异基因组移植术后第3天移植物内Tim3mRNA的表达明显升高,并且Tim3 mRNA的表达随着排斥反应的进行而显著升高(P0.01),于移植物完全排斥前达到最高峰,随后表达逐渐降低。移植术后受者外周血和脾脏内Tim3阳性细胞比例无明显变化(P0.05),引流淋巴结内Tim3+/CD4+比值轻度升高(P＜0.05),移植物内Tim3+/CD4+和Tim3+/CD8+比值显著升高(P0.01)。移植术后第3天和第6天引流淋巴结内Tim3+/CD4+比值差异无统计学意义(P0.05),而术后第6天移植物内Tim3+/CD4+和Tim3+/CD8+比值均显著高于第3天(P0.01)。结论移植物内Tim3 mRNA的表达与小鼠完全异基因心脏移植排斥反应的进展动态相关。异基因组受者移植物引流淋巴结和移植物内Tim3+细胞比例升高,以移植物内升高更为显著。目的观察Galectin-9对同种异体心脏移植物存活时间的影响,探讨Galectin-9激活Tim3-Tim3L通路在小鼠心脏移植模型中的免疫调节作用。方法建立小鼠同种异体心脏移植模型,分实验组和对照组。实验组受者术后连续7天给予稳定的Galectin-9蛋白,对照组给予PBS。观察心脏移植物存活时间。术后第7天获取实验组和对照组心脏移植物,病理切片观察排斥情况,免疫组化观察移植物内浸润CD4+和CD8+T细胞数量。实时定量RT-PCR检测移植物内Tim3,IFN-γ和IL-17mRNA的表达。流式细胞仪检测引流淋巴结和移植物内Tim3+细胞比例及外周血中Th1和Th17细胞比例。结果Galectin-9蛋白处理组心脏移植物平均存活时间为22.7天,对照组仅为7.2天,Galectin-9治疗组移植物存活时间显著延长(P0.05)。病理切片显示Galectin-9治疗组移植物排斥反应明显减轻,浸润的CD4+和CD8+T细胞明显减少。Galectin-9治疗后移植物内Tim3、IFN-γ和IL-17mRNA表达减少。引流淋巴结和移植物内Tim3+细胞比例降低,外周血中Th1和Th17细胞比例也降低。结论短期Galectin-9治疗能显著延长同种异体心脏移植物存活时间,其机制是减少移植物内淋巴细胞浸润,并通过触发Tim3+细胞程序性死亡而降低受者体内Th1和Th17细胞反应。目的观察阻断Tim4对小鼠心脏移植物存活时间的影响,探讨Tim4在Tim4-Timl通路诱导免疫耐受中的作用。方法建立小鼠同种异体心脏移植模型,给予受者抗Tim4抗体,观察其对移植物存活时间的影响,体外混合淋巴细胞培养检测抗Tim4抗体对同种反应性T细胞增殖的作用。比较Tim4基因缺陷和野生型心脏移植物在受体体内的存活时间,病理切片判断排斥情况,免疫荧光染色观察移植物内浸润淋巴细胞。实时定量RT-PCR检测移植物内免疫相关基因表达,流式细胞仪检测移植物浸润淋巴细胞亚型的变化。过继输注受者体内效应性T细胞和调节性T细胞给Babl/c SCID小鼠,观察其对皮肤移植物存活时间的影响。给予小剂量雷帕霉素观察其对Tim4基因缺陷的移植物存活时间的影响,并通过术前给予抗CD25抗体清除受者体内Treg明确Treg在本实验中的作用。结果抗Tim4抗体能抑制混合淋巴细胞培养体系中同种反应性T细胞增殖,延长移植物存活时间。供体Tim4基因缺陷明显减轻排斥反应,减少移植物内效应性T细胞浸润,增加调节性T细胞数量,从而显著延长移植物存活时间(P＜0.01)。Tim4基因缺陷能减少活化的效应性T细胞而增加Treg的数量,但是对效应性T细胞和Treg的功能无明显影响。联合小剂量雷帕霉素能诱导Tim4基因缺陷移植物长期存活。Tim4基因缺陷或/和小剂量雷帕霉素延长移植物存活时间依赖Treg的存在。结论Tim4在同种异体免疫反应中发挥重要的免疫调节作用,阻断Tim4-Timl通路能明显延长心脏移植物存活时间。
[Abstract]:Objective to establish a simple and effective method to separate infiltrating lymphocytes from cardiac allografts of mice, and to detect the subtypes of infiltrating lymphocytes in transplanted heart by flow cytometry.
Methods model of cervical heterotopic heart transplantation in mice, divided into experimental group (allograft group: donor Balb/c mice C57BL/6 mice as receptor) and control group (isograft group as donor and recipient C57BL/6 mice). 3 days after transplantation, 5 days and 7 days for heart transplantation, which will be part of the graft the pathological observation of rejection, graft and residual shear by type II collagenase digestion improved (250 U/ml, 30-40 min) digestion heart, then using Ficoll density gradient centrifugation (800g * 20 min) mononuclear cells were separated by calcium from the analysis of cell activity with fluorescence dye Indo-1, finally CD8, CD44 labeled antibodies of CD4, CD62L, Foxp3, flow cytometry staining and flow cytometry in the graft infiltrating lymphocyte subsets.
The number of mononuclear cells in the graft stable separation in 1 x 106, seventh days after operation the number reached a peak, separated from the number of mononuclear cells and the severity of rejection. Lymphocytes isolated from mononuclear cells of the ratio of (31.9 + 2.3)% and (95.1 + 2.1 to live%.) graft infiltrating T cells for the majority of effector T cells, the ratio of CD4+/CD8+ decreased gradually with the rejection.
Conclusion the method of single heart transplantation by collagenase digestion, using Ficoll density gradient centrifugation, reduce the damage of heart allografts infiltrating lymphocytes in plants, the cells can be further used for flow cytometric analysis of the phenotype, for direct detection of infiltration into the graft within the lymphocyte subtypes provides an effective and reliable method, is a worthy of study on the immunology method.
Objective to detect the expression changes of Tim3 mRNA in grafts and the expression of Tim3 in different parts of T lymphocytes in recipients, and to explore the relationship between them and acute rejection.
Methods mouse heart transplantation model, divided into experimental group (allograft group: donor Balb/c mice C57BL/6 mice as receptor) and control group (isograft group as donor and recipient C57BL/6 mice). After transplantation for 3 days, 5 days, 7 days and 9 days for the graft, using quantitative real-time RT-PCR detection of Tim3 mRNA expression in shift in plants. After 3 days and 6 days isolated from peripheral blood, spleen, lymph nodes, graft lymphocytes was detected by flow cytometry and Tim3 positive cells in CD4+ and CD8+T cells.
Results in isograft group the expression of Tim3 was very low. The expression of allogeneic transplantation after third days in the graft Tim3mRNA significantly increased the expression of Tim3 and mRNA with the rejection significantly increased (P0.01), to completely exclude the graft before reaching the peak, then the expression decreased gradually after transplantation by Tim3. The proportion of positive cells in peripheral blood and spleen had no significant change (P0.05), lymph node in Tim3+/CD4+ ratio increased slightly (P < 0.05), graft of Tim3+/CD4+ and Tim3+/CD8+ were significantly increased (P0.01). After transplantation for third days and sixth days of drainage Tim3+/CD4+ ratio difference in lymph nodes was not statistically significant (P0.05), and sixth days after surgery in the graft of Tim3+/CD4+ and Tim3+/CD8+ ratio were significantly higher than that of third days (P0.01).
Conclusion the expression of Tim3 mRNA is closely related to the progress of allograft heart transplantation rejection in allografts. The proportion of Tim3+ cells in the draining lymph nodes and the grafts in the allogeneic recipients is increased, and the increase in the graft is more significant.
Objective To observe the effect of Galectin-9 on the survival time of allograft cardiac allograft, and to explore the immunomodulatory effect of Galectin-9 activated Tim3-Tim3L pathway in mouse heart transplantation model.
Methods mouse cardiac allograft model, divided into experimental group and control group. The stability of Galectin-9 protein was administered for 7 consecutive days experimental group after operation, the control group was given PBS. to observe the cardiac allograft survival time. Seventh days after operation for the experimental group and the control group of cardiac allograft rejection, pathology observation, immunity histochemical observation of infiltrating CD4+ and CD8+T cells shift in plants. The number of real-time RT-PCR detection in the graft Tim3, IFN- expression and IL-17mRNA. Flow cytometry and lymph nodes in the graft ratio of Tim3+ cells and Th1 in peripheral blood and Th17 cell ratio.
Results Galectin-9 protein treatment group the mean survival time of cardiac allograft for 22.7 days, the control group was only 7.2 days, the Galectin-9 treatment group significantly prolong graft survival time (P0.05). The pathological sections showed that Galectin-9 treatment group significantly reduce graft rejection, infiltration of CD4+ and CD8+T were significantly decreased after treatment of.Galectin-9 graft in Tim3. Reduced expression of IFN- gamma and IL-17mRNA. The draining lymph node and Tim3+ cell ratio in plants to reduce the shift, Th1 in peripheral blood and Th17 cell ratio also decreased.
Conclusion short term Galectin-9 therapy can significantly prolong the survival time of allograft cardiac allograft, and its mechanism is to reduce lymphocyte infiltration in graft and reduce the Th1 and Th17 cell responses in vivo by triggering programmed cell death of Tim3+ cells.
Objective To observe the effect of blocking Tim4 on the survival time of cardiac allograft in mice and to explore the role of Tim4 in the induction of immune tolerance in the Tim4-Timl pathway.
Methods mouse cardiac allograft model, given by anti Tim4 antibody, to observe its effect on graft survival time, effect of mixed lymphocyte culture in vitro detection of anti Tim4 antibody on allogeneic T cell proliferation. Tim4 gene deficient and wild-type graft in recipients survival time and pathological judgment rejection, observed by immunofluorescence staining in the graft infiltrating lymphocytes. The expression of real time quantitative RT-PCR detection of shift of immune related genes in plants, the change of flow cytometry graft infiltrating lymphocyte subsets. Adoptive transfer recipients of effector T cells and regulatory T cells to Babl/c SCID mice to observe its effect the skin graft survival time. Give the effect of a small dose of rapamycin observed in Tim4 deficient graft survival time, and through the preoperative administration of anti CD25 The role of Treg in this experiment was scavenged in the body of the recipient.
The anti Tim4 antibody can inhibit the mixed lymphocyte culture system of alloreactive T cell proliferation, prolong graft survival. Donor Tim4 gene defects significantly reduce rejection, reduce the displacement effect of T cell infiltration in plants, increase the number of regulatory T cells, thereby significantly prolong graft survival time (P < 0.01).Tim4 gene the defect can reduce the activation of effector T cells and increase the number of Treg, but the effect on T cells and the function of Treg has no obvious effect. Combined with small dose of rapamycin could induce Tim4 gene defects in long-term graft survival of.Tim4 gene defect or / and small dose of rapamycin to prolong graft survival depends on the presence of Treg.
Conclusion Tim4 plays an important role in immunomodulating in allogeneic immune response, and blocking the Tim4-Timl pathway can significantly prolong the survival time of heart graft.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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