大鼠肺缺血再灌注损伤中肺泡细胞凋亡的动态观察

目的研究大鼠肺缺血再灌注损伤(IR)中肺泡细胞凋亡的动态过程及其与肺损伤的相关性。方法采用大鼠单肺原位热缺血再灌注模型。于缺血再灌注0min、30min、1h、2h,6h及12h采取肺组织及左房血标本,测定血氧分压、肺组织湿／干重比,并作光镜和透射电镜观察组织、细胞形态及亚显微结构,确定凋亡发生。TUNEL法测定肺组织中凋亡指数。台盼蓝活体染色评价细胞死亡程度,并结合同组凋亡水平间接推测细胞坏死水平。结果肺IR后,肺组织透射电镜观察可见肺泡Ⅱ型上皮细胞增多,并且不同程度出现形态学改变,细胞体积缩小变圆,细胞核呈多形性,核被膜外折或内陷,核固缩并边集在核膜下呈月牙状或腰带状,胞浆浓缩,胞浆内嗜锇性板层体减少、排空增多,细胞膜上微绒毛减少或消失,提示细胞凋亡发生,肺泡Ⅰ型上皮细胞则少有上述改变。单独缺血30min,肺泡细胞凋亡指数无增高。肺再灌注后0．5h起,肺泡细胞凋亡指数显著增高,再灌注2h时达到高峰。与细胞凋亡指数相比,坏死指数与肺功能损害的相关性更显著。结论肺IR后发生凋亡的主要是肺泡Ⅱ型上皮细胞。肺泡细胞凋亡指数于再灌注2h达到高峰。肺泡细胞坏死指数与肺功能损害的相关性较凋亡指数更显著。

Dynamic change of apoptosis of alveolar cells in ischemia-reperfusion induced pulmonary injury: an experimental study with rats

OBJECTIVE: To observe the dynamic changes of alveolar apoptosis in ischemia-reperfusion (IR) induced pulmonary injury, and to evaluate the roles of these two cell death styles, apoptosis and necrosis, in the progress of lung function deterioration in pulmonary IR injury. METHODS: Fifty-four Sprague-Dawley rats were made ischemia/reperfusion models by ischemia and reperfusion in situ in single lung. Thirty-six of the 54 rats in the experimental group were re-divided into 6 equal subgroups to undergo detection of partial pressure of oxygen (PaO2) of blood in left atrium, detection of lung tissue wet weight/dry weight ratio, histology of lung by light microscope, examination of ultrastructural changes of cells by transmission electron microscopy, and quantitative detection of apoptotic cells in the right middle lobe by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) 0 h, 0.5 h, 1 h, 2 h, 6 h, and 12 h respectively after the reperfusion (subgroups R0, R(0.5), R1, R2, R6, and R12). Another 18 rats in the experimental group were re-divided into 3 subgroups of 6 rats to undergo insertion of venous catheter into the main pulmonary artery via right ventricle to perfuse trypan blue so as to evaluate the cell death degree. The death index was observed under light microscope and the necrosis index was indirectly calculated by the equation: death index = apoptotic index + necrosis index. Thirty-six rats underwent sham operation. Twelve rats were used as preoperative blank controls. RESULTS: Proliferation of alveolar type II, but not alveolar type I cell, accompanied by ultrastructural morphological changes were seen 1 h, 2 h, and 6 h after reperfusion, the most prominently 2 h after reperfusion. Apoptotic index was elevated since 1 h after reperfusion, and peaked 2 h after reperfusion. Statistical analysis indicated that, compared with apoptotic index, the necrotic index was of more prominent correlation with blood oxygen partial pressure and wet/dry weight ratio. CONCLUSION: Alveolar apoptosis occurs in the early stage of reperfusion, and becomes the most prominent 2 h after reperfusion. Most apoptotic cells are alveolar type II cells. In the two styles of cell death in pulmonary IR injury, alveolar necrosis is more prominently correlated with progress of lung function deterioration.