【作者】 方泽民；

【导师】 陈忠华；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【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(P<0.01),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(P>0.05),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(P<0.01)。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(P>0.05),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(P<0.01)。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(P<0.05)。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。更多还原

【Abstract】 Objective To introduce a simple and efficient method for isolation of intragraft infiltrating lymphocytes and examine the subsets of lymphocytes by immunofluorescent staining and flow cytometer analysis.Methods The murine cardiac transplant model was built. Experimental group (Balb/c to C57BL/6) and control group (C57BL/6 to C57BL/6) were designed in this study. Cardiac grafts harvested on 3,5 and 7 days after operation respectively were cut into small pieces. These pieces were then digested with modified enzyme digestion singly using collagenaseⅡ(250 U/ml,30-40 min). Mononuclear cells were isolated by Ficoll density gradient centrifugation (800g×20 min). Ca2+fluorescence dye Indo-1 was used to analyze cell viability. Histological slices were made to observe the rejection. Subsets of lymphocytes were identified by flow cytometry after surface antigen and intracellular cytokine immunofluorescent staining.Results The harvested mononuclear cells were more than 1×106, which contained (31.9±2.3)%lymphocytes with viability of (95.1±2.1)%. The number of the mononuclear cells from the graft reached a peak on 7 days after transplantation and was closely correlated with the severity of rejection. Flow cytometry can analyze surface and intracellular antigens of the intragraft infiltrating lymphocytes at the same time. Most of the intragraft infiltrating lymphocytes were effector T cells. The ratio of CD4+/CD8+decreased gradually as the progression of rejection.Conclusion The modified method of isolating intragraft infiltrating lymphocytes with high viability is simple, reliable and efficient. It can be used widely in transplant immunology research.Objective To detect the changes of Tim3 expression in cardiac graft and T lymphocytes from different sites in mice heart-transplant recipients and explore the relationship of Tim3 expression level with acute rejection.Methods The murine cardiac transplant model was built. Experimental group (Balb/c to C57BL/6) and control group (C57BL/6 to C57BL/6) were designed in this study. Cardiac grafts were harvested on 3,5,7 and 9 days after operation respectively. Real-time quantitative RT-PCR was used to detect the local expression of Tim3 mRNA in graft. Lymphocytes were isolated from peripheral blood, spleen, draining lymph nodes and allograft on 3 days and 6 days after transplantation. The expression of Tim3 on T lymphocyte subsets CD4+and CD8+T cells was detected by flow cytometry.Results The expression of Tim3 was very low in isograft group. In allograft group, as the acute rejection occurred, the Tim3 expression increased significantly on 3 days after transplantation, which was related to the severity of rejection (P<0.01). It reached its peak on 7 days and then decreased gradually after the completely rejection. No significant increase of Tim3+cells was observed in peripheral blood and spleen after transplantation (P > 0.05). The expression of Tim3 on CD4+and CD8+of graft infiltrating T cells was obviously increased (P<0.01). While the ratio of Tim3+/CD4+had a slight elevation in draining lymph node (P<0.05). There was no obvious difference in Tim3+/CD4+ratio in draining lymph node between 3 days and 6 days (P> 0.05). But the ratios of Tim3+/CD4+ and Tim3+/CD8+in graft infiltrating T cells on 6 days were significantly (P<0.01) higher than that on 3 days.Conclusion The expression of Tim3 incresed in cardiac graft and T lymphocytes from draining lymph node and allograft after transplantation, which was correlated with the progression of acute rejection in mice.Objective To investigate the effect of Tim3 ligand (Galectin-9) on the survival of allograft and explore its immunomodulating role in an allogeneic heart transplant model.Methods Murine cardiac transplant model from BALB/c to C57BL/6 was built. The study was divided into experimental group and control group. Experimental group were administered with Galectin-9 for 7 days since day 1 posttransplant while control group with PBS. The complete cessation of heart beating was defined as the observation endpoint. The survival of allograft was observed in two groups. Histology and immunohistochemistry were performed to estimate the severity of rejection on 7 days posttransplant. Real-time quantitative RT-PCR was utilized to analyze the Tim3, IFN-γand IL-17 mRNA expression in allograft. The phenotype and cytokine profile of lymphocytes from peripheral blood, draining lymph nodes and allograft was analyzed by flow cytometry.Results The survival of allograft treated with Galectin-9 (mean survival time, MST= 22.7 days) was significantly prolonged compared with the control group (MST= 7.2 days), which was associated with reduced infiltration of CD4 and CD8 lymphocytes in allograft. Galectin-9 decreased the expression of Tim3, IFN-γand IL-17 mRNA in allograft notably. The proportion of Tim3 positive cells in draining lymph nodes and allograft and Thl and Th17 positive cell in peripheral blood was obviously lower than the control group.Conclusion A short-course administration of Galectin-9 significantly prolonged the survival of fully allogeneic cardiac allografts, which was associated with the suppression of Th1 and Th17 immune responses.Objective To study the effect of Tim4 blockade on the survival of cardiac allograft and explore the role of Tim4 in Tim4-Tim1 pathway in inducing immune tolerance.Methods Murine cardiac transplant model from BALB/c to C57BL/6 was built. The survival was compared between the recipients treated with anti-Tim4 Ab and PBS. MLC was used to assess the effect of anti-Tim4 Ab on cell proliferation. The survival of Tim4-/-and wild-type graft in recipients was observed. Histology was performed to estimate the severity of rejection. Immunofluorescent staining was carried out for detecting lymphocytes in allograft. The expression of immune-related genes was determined by Real-time quantitative RT-PCR. The subsets of intragraft infiltrating lymphocytes were analyzed by flow cytometry. Effector T cells and Tregs from the recipients were sorted by FACS. These cells were then transfused into Balb/c SCID mice followed by implantation of skin allograft. The rejection and survival of the allograft were observed. The effect of low dose rapamycin on the survival of Tim4-/- allograft was studied. The role of Tregs in the model was evaluated by administering anti-CD25 mAb before transplantation.Results Recipients conditioned with anti-Tim4 mAb exhibited prolonged cardiac allograft survival with suppressing the T cell proliferation in MLC. The survival of Tim4-/- allograft was significantly prolonged compared with wild-type group. The prolonged survival time was associated with reduced lymphocytes and increased Treg in allografts. Strikingly, when combined with a low dose of rapamycin, Tim4-/- heart allografts survived indefinitely. The absence of Tim4 led to the decreased recruitment, but not decreased effect, in the effector T cells compartment. Similarly, it increased recruitment, but not increased function, in the Treg compartment. Moreover, depletion of Treg with anti-CD25 Ab completely abrogated the tolerogenic effects of Tim4 deficiency alone or when combined with rapamycin.Conclusion A blockade of the Tim4-Tim1 axis improves allograft survival as a monotherapy and synergizes with low-dose rapamycin to induce complete transplantation tolerance by enhancing immunoregulation.更多还原