非协调性异种气管移植的实验研究

Experimental research of discordant tracheal xenotransplantation

目的探索异种气管移植免疫排斥反应特点,为解决供体气管来源提供新方向,并为研究肺移植继发的气道阻塞性疾病(OAD)建立理想的动物模型。方法建立SD大鼠颈部肌肉瓣包裹移植气管模型,以深低温冻储同种异体气管移植为对照,通过组织化学检查,免疫荧光检查,流式细胞术等方法,观察冷冻与非冷冻豚鼠—大鼠非协调性异种气管移植的成活情况,分析其免疫排斥反应的特点和机制。结果颈部肌肉瓣包裹深低温冻储同种异体SD大鼠长期存活。豚鼠—大鼠冷冻异种气管移植最长成活14 d,平均(13.2±0.75)d;新鲜异种气管移植最长成活9 d,平均(8.0±1.09)d。组织学检查,异体移植气管基本正常,气管通畅度大于80%。异种移植气管呈急性排斥反应表现,移植物大量嗜酸粒细胞,淋巴细胞,单核巨嗜细胞浸润;受体IgM,IgG,C3沉积;外周血CD4+T、CD8+T淋巴细胞明显升高;黏膜上皮剥脱,软骨失去活性;气管通畅度小于50%。以上表现随时间延长而加重,冷冻组弱于非冷冻组。结论细胞免疫反应参与的体液免疫反应为主的急性排斥反应是豚鼠—大鼠非协调性异种气管移植免疫反应特点。深低温冻储消减供体抗原,在一定范围内延长异种移植物成活时间。

Objective To investigate the immunological rejection mechanism of tracheal xenotransplantation and xenografts as potential sources of trachea. Methods On SD rat model, a xenotransplanted tracheal from the guinea pig was established by wrapping it in the cervical muscles in situ. It was divided into cryopreserved group and uncryopreserved group. Under the examinations with histochemistry, immunofluorescence （IFL） and flow cytometry （FCM） techniques, the pathomorphological characteristics of the tracheal xenografts and the immunological rejection mechanism were evaluated. Results The tracheal aUotransplantation with cryopreserved grafts wrapped by neck muscles was survived for a longer period. Histological examination revealed normal appearance of the aUografts. The tracheal grafts potency was above 80%. However, cryopreserved tracheal xenografts of the guinea pig-to-rat maintained vitality for 14 days in maximum and 13.2 days on average, while the fresh tracheal xenografts only for 9 days in maximum, and 8 days on average. Acute rejection occurred in the tracheal xenotransplantation. A marked mononuclear-macrophage cellular infiltration mixed with eosinophils and lymphocyte was seen in the xenografts. Antibody （IgM, IgG） and complement （C3） deposition were also obviously detected by IFL in the xenografts. CD4^＋ T ceils and CD8^＋ T ceils increased significantly in the vascular circulation. In all of the xenografts, complete loss of tracheal epithelium was associated with cartilage necrosis. The grafts patency was below 50%. This performance deteriorated with extended time periods. The fresh xenografts performed significantly worse than the cryopreserved xenografts. Conclusions Acute rejection, caused by humoral immune reaction mainly integrated with cellular immunity, is the most notable characteristics in the guinea pig-to-rat tracheal xenotransplantation in situ. Cryopresrvation can potentially reduce the antigenicity. The low antigenicity may inhibit the immunologic reaction relatively, so that prolonged survival of discordant cryopreserved tracheal xenografts could be achieved.