摘要
目的建立小鼠原位左肺移植模型,探究抗CD3单抗缓解肺移植急性排斥损伤的作用机制,为其在临床肺移植的应用提供理论依据。方法选取SPF级野生型BALB/c和C57BL/6小鼠,构建原位左肺移植模型,设置同种同型对照组(C57BL/6→C57BL/6,6只)、同种异型对照组(BALB/c→C57BL/6,6只),同种异型单抗处理组(BALB/c→C57BL/6,4只)。术后第2~6天及第9天每日腹腔注射50g抗CD3单抗。采用苏木精-伊红、马松染色以及CD3/髓过氧化物酶(MPO)免疫组化染色,观察各组移植肺T淋巴细胞和中性粒细胞浸润分布情况,进行急性排斥病理评分;实时荧光定量RT-PCR检测移植肺组织转录因子FoxP3和细胞因子IL-17A、IFN-γ的mRNA表达水平;流式细胞术检测受体小鼠脾脏中FoxP3+调节性T细胞(Treg)占CD4+T细胞的比例。结果移植术后10天,同种同型组移植肺外观呈淡红色,柔软有弹性,病理学检测未见明显的炎症细胞浸润和组织损伤;同种异型对照组移植肺外观呈绛紫色,硬度增加;同种异型单抗处理组外观淡红柔软,与同种异型对照组比较,淋巴细胞、中性粒细胞浸润显著减少,急性排斥损伤病理评分降低。实时荧光定量RT-PCR结果显示,与同种同型组相比,同种异型组移植肺中IL-17A和IFN-γmRNA的表达水平升高;抗CD3单抗可降低移植肺中IL-17A与IFN-γmRNA表达水平,升高FoxP3mRNA表达水平。流式细胞术结果显示,与同种同型、同种异型对照组相比,单抗处理组小鼠脾脏中Treg占CD4+T细胞的比例显著升高。结论抗CD3单抗可诱导CD4+FoxP3+Treg的分化并缓解肺移植急性排斥损伤。
Objective By using mouse orthotopic lung transplant model, we investigated the immune mechanisms of anti-CD3 induced lung allograft protection. Our study intends to further dissect the features of lung transplant immunology and to provide a novel therapeutic insight for the clinical application of anti-CD3 mAbs after lung transplantation. Methods Murine orthotopic allogeneic lung transplants were performed in C57BL/6 wild type(WT) mice using major histocompatibility complex(MHC) fully mismatched BALB/c donors. Syngeneic transplants were also performed in WT C57BL/6 mice using C57BL/6 donors. For immunosuppressive therapy, allograft recipients received 50g dose of anti-CD3 by intraperitoneal injection on days 2, 3, 4, 5, 6 and 9 post-operation(n=4). At day 10, histopathologic characteristics and rejection status of the pulmonary grafts were assessed. The severity of acute rejection was graded by the pathological score, and T cell and neutrophil infiltration in the pulmonary grafts was evaluated by immunohistochemical(IHC) staining for CD3 and myeloperoxidase(MPO) respectively. Real-time RT-PCR was performed for FoxP3, IL-17A and IFN-γ expression in the pulmonary grafts. The percentage of FoxP3+ Treg in total CD4+ T lymphocytes from the recipient spleens was analyzed by FACS. Results 10 days after transplantation, histopathologic examination demonstrated that there is no apparent acute rejection observed in the pulmonary isografts, whereas allografts from untreated recipients have marked inflammatory cell infiltration and pulmonary parenchyma lesion. IHC staining for CD3 and MPO showed that the allograft-infiltrating cells of perivascular layers are mainly T lymphocytes, and the cells around the small airways are mostly neutrophils. Anti-CD3 treatment significantly alleviated the acute rejection of pulmonary allografts, when compared with the untreated group. Real-time RT-PCR showed that the expression levels of IL-17A and IFN-γ in allografts were markedly elevated compared to those in isografts, and anti-CD3 inc
作者
梁凡
陈荣卷
李燕燕
丁跃中
许江南
Liang Fan;Chen Rongjuan;Li Yanyan;Ding Yuezhong;Xu Jiangnan(Department of Immunology, Capital Medical University, Beijing 100069, China)
出处
《中华胸心血管外科杂志》
CSCD
北大核心
2019年第5期307-311,共5页
Chinese Journal of Thoracic and Cardiovascular Surgery
基金
国家自然科学基金(81770092).
关键词
小鼠原位肺移植
急性排斥
抗CD3单抗
调节性T细胞
Orthotopic lung transplantation
Acute rejection
Anti-CD3 monoclonal antibodies(Anti-CD3 mAbs)
Regulatory T(Treg) cells
