肺保护液肺动脉灌注的实验研究
摘要
目的 建立一种兔在体的左肺缺血、肺动脉保存液灌注、低温保存、再灌注损伤动物模型,用以比较、筛选和鉴定不同肺保护液对在体肺缺血冷藏再灌注后肺组织结构和功能的影响,最大限度地减少非实验因素对肺保存液保存效果的干扰,以优选肺保护液,为临床肺保护液的开发提供实验工具。
方法 新西兰白兔50只,体重2500～3500g。其中10只用于预实验,建立并逐步改进兔在体左肺缺血、肺动脉灌注、冷藏、再灌注损伤动物模型。40只新西兰白兔用于正式实验。动物随机分为A、B、C、D四组,每组10只。经左侧开胸,掀开胸壁,游离左上肺动脉,穿刺置管,切除左肺上叶,经左心房置管入左肺下静脉。A组(对照组):单纯阻断左肺门,左肺不灌注肺保护液,阻断左肺门后直接将左肺下叶放入特制的肺保存器内10℃冷藏2小时,移去肺保存器,再灌注2小时;B组(LPD组):左肺门阻断后经肺动脉灌注4℃ LPD肺保护液,经肺静脉内置管引流,余同对照组;C组(抑肽酶组):灌注液为含抑肽酶的LPD液,余同LPD组。D组(GBE组):灌注液为含GBE的LPD液,余同LPD组。分别于肺门阻断前、再灌注5分钟、120分钟取肺静脉血,行血气分析,测定正常、缺血后及再灌注损伤后肺静脉血氧含量,以评价保存后肺氧合功能。取左肺上叶正常组织及肺门开放前、开放后120分钟取左肺下叶肺组织,10%中性甲醛固定,H-E染色观察组织学改变,免疫组化染色观察肺组织P-选择素及细胞间粘附分子-1基因mRNA表达的情况。将左肺组织取标本后余肺用吸水纸拭干,称重得标本湿重,将标本置入72℃烤箱内,烘烤72小时后称重记录为干重,计算出肺湿/干重比,检查肺水肿情况。
结果 在预实验阶段,10只动物因出血死亡,或因出血导致的后续操作无法进行而致失败5只,成功率50%。经过改进后在正
Objective To establish a rabbit in situ left lung preservation model undergoing ischemia, pulmonary infusion with lung preservation solution, cold storage and reperfusion which can be used to compare, identify lung preservation solution and can minimize the disturbance of non-experimental factors for the study of effectiveness of lung slush solution. Methods Fifty New Zealand white rabbits were selected as experimental subjects, weight ranging from 2500~3500g. Ten New Zealand white rabbits were used at the pre-experimental stage, in order to establish and optimize the rabbit in situ left lung ischemia, pulmonary infusion, cold storage and reperfusion model (rabbit in situ lung preservation model). Forty New Zealand white rabbits were used at the formal experimental stage. By left thoracotomy, left superior pulmonary artery was separated and was inserted into a catheter and then left upper lobe was removed. Another catheter was inserted into the left inferior pulmonary vein via left atrial wall. Both catheters were connected to sensors to detected pressure. After left lung root was clamped, lung flush solution was perfused through the catheter in the left superior pulmonary artery and was drained via the catheter in the left inferior pulmonary vein. The left lower lung lobe was put and stored in a specially made lung container at 10 centigrade for 2 hours. Thereafter, the lung root was unclamped for 2 hours. After samples were collected, the subjects were executed by euthanasia. Results In the pre-experimental stage, 5 rabbits out of 10 failed to finish the model making due to hemorrhage, with the successful rate of 50%. With the innovated technique, in the formal experimental stage, all the 40
rabbit models were accomplished successfully. The physiological parameters, such as blood pressure, heart rate and body temperature at pre-thoractomy (89.8 ± 11.9mmHg,> 213.4 + 34.7bpm^ 39.06 + 0.12 °C ), pre-clamping of the lung hilus(88.6 + 5.5 mmHg, 216.0 + 27.7bpm,39.06 + 0.23"C)>30 minutes after clamping ofthe lung hilus( 91.6 + 8.3 mmHg > 211.5 + 27.5bpm , 39.04 + 0.28 °C ) , pre-unclamping the lung hilus (90.5 + 8.8 mmHgx 2 12.1 + 25.3bpm> 39.05±0.32°C K post-unclamping the lung hilus ( 90.8 + 8.2 mmHg, 211.6 + 30.9bpn^ 39.04 + 0.32°C ), before end of experiment ( 91.0 + 5.9mmHg, 210.1 ± 27.8bpm > 38.97 +0.13 °C ) were all stable and there was no significant difference among different time points. All the specimen were obtained successfully. Conclusion Rabbit in situ lung preservation model, as an ideal model to study the effectiveness of lung slush solution, is simple, physiological, stable, safe and practical, which can avoid the disturbance of many non-experimental factors. It can also be fed after end of experiment to make chronic animal model to investigate the long term effect on lung tissue after lung ischemia, cold storage and reperfusion.
方法 新西兰白兔50只,体重2500～3500g。其中10只用于预实验,建立并逐步改进兔在体左肺缺血、肺动脉灌注、冷藏、再灌注损伤动物模型。40只新西兰白兔用于正式实验。动物随机分为A、B、C、D四组,每组10只。经左侧开胸,掀开胸壁,游离左上肺动脉,穿刺置管,切除左肺上叶,经左心房置管入左肺下静脉。A组(对照组):单纯阻断左肺门,左肺不灌注肺保护液,阻断左肺门后直接将左肺下叶放入特制的肺保存器内10℃冷藏2小时,移去肺保存器,再灌注2小时;B组(LPD组):左肺门阻断后经肺动脉灌注4℃ LPD肺保护液,经肺静脉内置管引流,余同对照组;C组(抑肽酶组):灌注液为含抑肽酶的LPD液,余同LPD组。D组(GBE组):灌注液为含GBE的LPD液,余同LPD组。分别于肺门阻断前、再灌注5分钟、120分钟取肺静脉血,行血气分析,测定正常、缺血后及再灌注损伤后肺静脉血氧含量,以评价保存后肺氧合功能。取左肺上叶正常组织及肺门开放前、开放后120分钟取左肺下叶肺组织,10%中性甲醛固定,H-E染色观察组织学改变,免疫组化染色观察肺组织P-选择素及细胞间粘附分子-1基因mRNA表达的情况。将左肺组织取标本后余肺用吸水纸拭干,称重得标本湿重,将标本置入72℃烤箱内,烘烤72小时后称重记录为干重,计算出肺湿/干重比,检查肺水肿情况。
结果 在预实验阶段,10只动物因出血死亡,或因出血导致的后续操作无法进行而致失败5只,成功率50%。经过改进后在正
Objective To establish a rabbit in situ left lung preservation model undergoing ischemia, pulmonary infusion with lung preservation solution, cold storage and reperfusion which can be used to compare, identify lung preservation solution and can minimize the disturbance of non-experimental factors for the study of effectiveness of lung slush solution. Methods Fifty New Zealand white rabbits were selected as experimental subjects, weight ranging from 2500~3500g. Ten New Zealand white rabbits were used at the pre-experimental stage, in order to establish and optimize the rabbit in situ left lung ischemia, pulmonary infusion, cold storage and reperfusion model (rabbit in situ lung preservation model). Forty New Zealand white rabbits were used at the formal experimental stage. By left thoracotomy, left superior pulmonary artery was separated and was inserted into a catheter and then left upper lobe was removed. Another catheter was inserted into the left inferior pulmonary vein via left atrial wall. Both catheters were connected to sensors to detected pressure. After left lung root was clamped, lung flush solution was perfused through the catheter in the left superior pulmonary artery and was drained via the catheter in the left inferior pulmonary vein. The left lower lung lobe was put and stored in a specially made lung container at 10 centigrade for 2 hours. Thereafter, the lung root was unclamped for 2 hours. After samples were collected, the subjects were executed by euthanasia. Results In the pre-experimental stage, 5 rabbits out of 10 failed to finish the model making due to hemorrhage, with the successful rate of 50%. With the innovated technique, in the formal experimental stage, all the 40
rabbit models were accomplished successfully. The physiological parameters, such as blood pressure, heart rate and body temperature at pre-thoractomy (89.8 ± 11.9mmHg,> 213.4 + 34.7bpm^ 39.06 + 0.12 °C ), pre-clamping of the lung hilus(88.6 + 5.5 mmHg, 216.0 + 27.7bpm,39.06 + 0.23"C)>30 minutes after clamping ofthe lung hilus( 91.6 + 8.3 mmHg > 211.5 + 27.5bpm , 39.04 + 0.28 °C ) , pre-unclamping the lung hilus (90.5 + 8.8 mmHgx 2 12.1 + 25.3bpm> 39.05±0.32°C K post-unclamping the lung hilus ( 90.8 + 8.2 mmHg, 211.6 + 30.9bpn^ 39.04 + 0.32°C ), before end of experiment ( 91.0 + 5.9mmHg, 210.1 ± 27.8bpm > 38.97 +0.13 °C ) were all stable and there was no significant difference among different time points. All the specimen were obtained successfully. Conclusion Rabbit in situ lung preservation model, as an ideal model to study the effectiveness of lung slush solution, is simple, physiological, stable, safe and practical, which can avoid the disturbance of many non-experimental factors. It can also be fed after end of experiment to make chronic animal model to investigate the long term effect on lung tissue after lung ischemia, cold storage and reperfusion.
引文
1 The Toronto Lung Transplant Group. Unilateral lung transplantation for pulmonary fibrosis. N Engl J Med,1986,314:1140-1145.
2 Copper JD, Pearson FG, Patterson GA, et al. Techniques for successful lung transplantation in humans. J Thorac Cardiovasc Surg, 1987,93:182-198.
3 陈静瑜.第一届全国肺移植高级研讨会会议简况.中华医学杂志,2004,84(19):1661.
4 Connaughton PG, Baohuth JJ, Leuis FJ. Lung ischemia up to six hours: Influence of cooling in situ on subsequent pulmonary function. Disease Chest, 1962,41(3): 404-408.
5 苏雷,魏秀芹,陈玉平,等.犬移植肺缺血再灌注实验动物模型的建立.中国胸心血管外科临床杂志,1999,6(2):109-111.
6 Takatoshi M, Akira K, Kazuya N, et al. Simplified rat lung transplantation using a cuff technique. Transplantation proceedings,1989,21:2601-2602.
7 许后效,金秀兰。雪貂和旱獭作为胃癌病因研究的实验模型。环境科学进展,1995,3(6):58-63.
8 蒋志华,杨辰垣.大鼠同种异体左肺原位移植模型的建立.中华实验外科杂志,1999,16(2):185-186.
9 Divisi D, Montagna P, Jegaden O, et al. Lung transplantation by continuous perfusion in an experimental auto-transplant animal model. Thorac Cardiovasc Surg,2002,50(5): 301-305.
10 Eren S, Esme H, Balci AE, et al. the effect of aprotinin on ischemia-reperfusion injury in an in situ normothermic ischemic lung model. Eur J Cardiothorac Surg,2003,23(1): 60-65.
11 潘铁文,吕随峰,孙耀昌.三袖套管法大鼠肺移植.中华器官移植杂志,1998,19(2):84-86.
12 夏穗生,田霞,卢永顺。新一代喷涂型福爱乐医用胶(基础研究)。临床外科杂志,2003,11(2):120-121。
13 夏穗生,田霞,卢永顺。新一代喷涂型福爱乐医用胶(临床应用)。临床外科杂志,2003,11(3):190-191.
14 宋继明,许洪春。福爱乐医用胶在支气管胸膜瘘中的临床应用。临床外科杂志,2004,12(11):711-712。
15 马海涛,秦涌,何靖康。福爱乐医用胶在普胸外科手术中的应用。临床外科杂志,2003,11(6):6441-442。
2 Copper JD, Pearson FG, Patterson GA, et al. Techniques for successful lung transplantation in humans. J Thorac Cardiovasc Surg, 1987,93:182-198.
3 陈静瑜.第一届全国肺移植高级研讨会会议简况.中华医学杂志,2004,84(19):1661.
4 Connaughton PG, Baohuth JJ, Leuis FJ. Lung ischemia up to six hours: Influence of cooling in situ on subsequent pulmonary function. Disease Chest, 1962,41(3): 404-408.
5 苏雷,魏秀芹,陈玉平,等.犬移植肺缺血再灌注实验动物模型的建立.中国胸心血管外科临床杂志,1999,6(2):109-111.
6 Takatoshi M, Akira K, Kazuya N, et al. Simplified rat lung transplantation using a cuff technique. Transplantation proceedings,1989,21:2601-2602.
7 许后效,金秀兰。雪貂和旱獭作为胃癌病因研究的实验模型。环境科学进展,1995,3(6):58-63.
8 蒋志华,杨辰垣.大鼠同种异体左肺原位移植模型的建立.中华实验外科杂志,1999,16(2):185-186.
9 Divisi D, Montagna P, Jegaden O, et al. Lung transplantation by continuous perfusion in an experimental auto-transplant animal model. Thorac Cardiovasc Surg,2002,50(5): 301-305.
10 Eren S, Esme H, Balci AE, et al. the effect of aprotinin on ischemia-reperfusion injury in an in situ normothermic ischemic lung model. Eur J Cardiothorac Surg,2003,23(1): 60-65.
11 潘铁文,吕随峰,孙耀昌.三袖套管法大鼠肺移植.中华器官移植杂志,1998,19(2):84-86.
12 夏穗生,田霞,卢永顺。新一代喷涂型福爱乐医用胶(基础研究)。临床外科杂志,2003,11(2):120-121。
13 夏穗生,田霞,卢永顺。新一代喷涂型福爱乐医用胶(临床应用)。临床外科杂志,2003,11(3):190-191.
14 宋继明,许洪春。福爱乐医用胶在支气管胸膜瘘中的临床应用。临床外科杂志,2004,12(11):711-712。
15 马海涛,秦涌,何靖康。福爱乐医用胶在普胸外科手术中的应用。临床外科杂志,2003,11(6):6441-442。