大鼠肝移植术后肝脏T淋巴细胞凋亡与免疫耐受的关系

Relationship between immune tolerance and hepatic T lymphocytes apoptosis after liver transplantation in rats

目的探讨肝移植术后肝组织T淋巴细胞凋亡和肝移植免疫耐受之间的关系。方法采用Kamada二袖套法建立Wistar→Sprague-Dawley(SD)原位肝移植(OLT)大鼠模型。实验动物随机分为3组,每组各6只。A组:空白对照组,不作任何处理,采用SD大鼠;B组:免疫排斥组,Wistar大鼠为供体,SD大鼠为受体,行OLT;C组:免疫耐受组,Wistar大鼠为供体、SD大鼠为受体,行OLT,术前1周胸腺内注射F蛋白0.4mg,建立稳定的移植耐受大鼠模型。A组立即处死大鼠,B组和C组分别于术后7d、100d处死大鼠取肝组织,分别取各组大鼠肝组织标本进行冰冻切片,应用原位末端脱氧核苷酸转移酶标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)在荧光显微镜下检测肝移植术后肝脏T淋巴细胞的凋亡情况。各组样本另取一张切片行常规苏木素-伊红(HE)染色在光学显微镜下观察,与TUNEL荧光染色法作对比观察。结果光学显微镜下,B组可见中、重度免疫排斥反应表现,C组肝组织细胞间的淋巴细胞浸润较B组大大减少,稍多于A组。荧光显微镜下,A组TUNEL切片可见零星散在的凋亡细胞,C组可见大量散在或密集分布的凋亡细胞,B组的凋亡细胞数远较C组减少,但仍多于A组。A组、B组、C组大鼠肝组织内浸润T淋巴细胞的凋亡指数(apoptosisindex,AI)分别为(8.83±0.43)%、(11.32±1.29)%和(19.00±1.96)%,两两比较差异有统计学意义(P<0.05～0.01)。结论免疫耐受移植物内浸润的T淋巴细胞凋亡明显增高,浸润的T淋巴细胞凋亡受阻可能会阻碍免疫耐受的发生,引起排斥反应。

Objective To analyze the relationship between apoptosis of hepatic T lymphocytes and immune tolerance after liver transplantation. Methods The rat model of orthotopic liver transplantation （OLT） from Wistar rats to Sprague-Dawley （SD） rats was performed by Kamada＇s two-cuff technique. Then the rats were randomly divided into three groups with 6 rats in each group. Group A ： blank control group （ SD rats were used without any treatment）. Group B： immune rejection group （OLT was performed from Wistar rats to SD rats）. Group C： immune tolerance group （OLT was performed from Wistar rats to SD rats. F protein （0. 4 rag） was injected into thymus at the 7^th day before transplantation to establish a stable rat model of transplant immtme tolerance）. Rats in Group A were executed immediately. Rats in Group B and C were executed at 7 d and 100 d after transplantation to obtain the liver tissues. Liver tissues in each group were obtained and observed by frozen section. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） was used todetect apop-tosis of T lymphocytes after liver transplantation under fluorescence microscope. Another section in each sample was stained by routine hematoxylin-eosin （HE） and observed under the microscope to compare with TUNEL methods. Results In the microscope, the expression of moderate and severe rejection was observed in Group B. The infiltrated lymphocytes in hepatic cells in Group C were much less than those in Group B, but a little more than those in Group A. In fluorescence microscope, apoptotic cells scattered sporadically in TUNEL sections of Group A. In Group C, apoptotic cells interspersed in a large number or densely distributed. The number of apoptotic cells in Group B was much less than that in Group C, but still more than that in Group A. Apoptosis index （AI） of infiltrated T lymphocytes in hepatic tissues of Group A, B and C were respectively（8. 83 ± 0. 43 ）%, （11.32 ± 1.29）% and （19. 00 ±1.96 ）%, in which there were significant difference （P 〈 0. 05 - 0. 01 ）. Conclusions In the graft with immune tolerance, the apoptosis of infiltrated T lymphocytes increases significantly. Blocking the apoptosis of infiltrated T lymphocytes may suffocate occurrence of immune tolerance and cause rejection.