抑肽酶对离体模拟肺移植兔供体肺功能及细胞凋亡的影响

Effect of aprotinin on improving graft function and inhibiting apoptosis of pulmonary cells of lung inplant after lung transplantation

目的探讨低钾右旋糖酐液(LPD)中加入抑肽酶对供体肺功能的保护作用及供体肺细胞凋亡的影响。方法24只健康纯种新西兰大白兔随机分为两组,A组用LPD液(70ml/kg)经肺动脉灌注供体肺,B组用含150kU/ml抑肽酶的LPD液(70ml/kg)灌注供体肺,4℃保存16h,应用肺移植动物模型模拟肺移植过程1h。循环灌注期间每隔10min记录肺动脉压(PaP)、气道峰压(PawP),并检测供体肺血气标本(PaO2、PaCO2),计算肺泡-动脉血氧差(A-aDO2)、肺顺应性(CL)。光镜、电镜观察各组供体肺移植前后的组织变化;TUNEL-Fluorescein及PI双染色法定量观察各组供体肺移植前后细胞凋亡变化,分析细胞凋亡与供体肺功能的关系。结果再灌注早期(前20min)两组供体肺氧合能力无差异,再灌注30min后各时段,A组PaO2低于B组(54.42～118.87mmHgvs112.33～183.73mmHg,P<0.01);B组供体肺在整个灌注过程中能保持良好的氧合能力(PaO2≥112.33mmHg)。再灌注末期,A组PaCO2及含水量均明显高于B组(50.96mmHgvs41.42mmHg,P<0.01;88.90%vs82.48%,P<0.001),B组供体肺的PaP、PawP、CL则明显优于A组。光镜及电镜观察见A组供体肺细胞结构破坏,间质水肿,而B组则基本保持正常结构。两组供体肺移植后均出现明显的细胞凋亡,且A组凋亡指数明显高于B组(24.67%vs15.50%,P<0.01)。供体肺凋亡指数与PaO2、CL呈负相关(r=-0.895,r=-0.683,P<0.001)。结论LPD液中加入抑肽酶可提高移植术后供体肺功能,抑制移植后早期供体肺细胞的凋亡;细胞凋亡可能是供体肺移植术后早期肺功能减低的原因之一。

Objective To study the effect of aprotinin on improving graft function and inhibiting cell apoptosis of cells of donor lung after transplantation and to in vestigate the relation between post-transplant lung function and apoptosis. Methods On an isolated, whole blood-perfused, ventilated rabbit lung transplantation model, 24 rabbits were divided into two groups： in group A the donor lung block was flushed with LPD solution through the pulmonary artery, and in group B aprotinin in 150KIU/ml was added to the pulmonary flushing fluid. Oxygenation capacity （PaO2, PaCO2） and pulmonary compliance （PAP, PawP, CL） were continuously monitored during the one-hour cyclic perfusion. The structural and ultrastructural changes in the donor lung were examined before and after transplantation. Terminal deoxynuclcotidyl transferase-uridine nuclcotide end-labeling （TUNEL-Fluorescein） and propidium iodide （PI） double staining techniques were used to detect apoptosis of donor lung before and after transplantation. Results The donor lung in group B had significantly better oxygenation capacity during 60 minutes of reperfusion （PaO2 〉112. 33mmHg）. PaO2 of group B was significantly higher than those of group A after 30 minutes of reperfusion （54. 42-118. 87mmHg vs 112. 33-183. 73mmHg, P〈0.01）. PaCO2 in group A was significantly higher than that in group B （50. 96mmHg vs 41.42mmHg, P〈0.01）. PAP and PawP in group A were higher than that in group B after 20min and 30min of reperfusion （P〈0. 001）. CL of group B was higher than that of group A （0. 156 vs 0. 120cm^3 · cmH2O^-1 , P〈0.01）. Electron microscopy revealed that ultrastructure of donor lung in group B was well preserved after 60min of reperfusion. TUNEL-Fluorescein and PI double staining revealed that almost no evidence of apoptosis was found in specimens after cold ischemic periods. Significant increase in the numbers of cells undergoing apoptosis were observed after graft reperfusion. And there were significantly more apoptotic ceils in groups A （24. 67%） compared with groups B （15.50%） after transplantation （P〈0. 01）. The percentage of apoptosis ceils were inversely correlated with post-transplant donor lung function （r=-0. 895, P〈0. 001）. Conclusion The addition of aprotinin to LPD solution may improve donor lung function by inhibiting apoptosis in the early reperfusion period. The apoptosis might contribute to ischemia-reperfusion injury during the early phase of graft reperfusion and be responsible for severe organ dysfunction.