犬肺移植中供体肺NO、MDA、SOD与形态学变化的相关性研究
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摘要
研究目的
在分析ECS及MECS的基础上修改配方成分自制IECS；探讨犬肺
低温灌洗、离体保存过程中随着冷缺血时间推移NO、MDA、SOD的
变化规律；探讨分子水平肺组织活性改变与形态学变化的相互关系以及
体视学量化原理在供体肺研究中的可适用性。
材料和方法
12只雄性健康杂种犬随机分入供体组和受体组，供体组之供肺以
灌洗前为对照组；灌洗后低温保存期间不同时间点为实验观察组。麻醉
生效后，供体组：开胸后经肺动脉插管注入PGE_1400μg，采用低温单次
原位顺行法灌洗4℃ IECS 1500ml，压力控制在3～4kPa。灌洗完毕后
完整摘取心肺，一侧肺作为供肺移植，另一侧肺离体低温（4℃）保存，
测试、观察不同时间点肺组织分子生化和形态学改变，包括：肺组织
W/D；制备匀浆，测定肺组织内NO、MDA和SOD浓度；肺组织形态
学变化的定性观察及描述；用方网测试系统体视学量化原理测定肺泡上
皮细胞核/质Vv、Sv、Rsv。受体组：开胸后行单侧全肺切除并修整残
端，待移植肺残端修整后与之吻合；移植前、中、后监测血流动力学及
呼吸各项参数指标。
结 果
实验组与对照组相比较：W/D，NO、SOD和MDA含量，肺泡内
出血分级，肺泡上皮细胞核/质Vv、Sv、Rsv随低温保存时间变化无显
著性差异（P＞0.05）；但随时间延长NO、SOD、Vv、Sv均呈下降趋势，
而MDA、W／D、RSV却略有上升趋势。保存期间细小支气管上皮细胞
和血管内皮细胞结构正常，肺组织、肺泡内出血变化不明显，主要改变
是组织水肿。
结 论
1艰据犬肺离体低温保存后肺组织Wto，NO、SOD和 MDA含量，
肺泡内出血分级，肺泡上皮细胞核／质VV、SV、RSV随时间延长变化的
特点，可见肺组织病理形态学改变略滞后于OFR变化，随着保存时间
延长肺组织水肿渐趋明显。
2在本实验条件下所采用的IECS进行犬肺灌洗、离体保存4h之内
肺血管内皮细胞无明显损伤，PGE；具有保护肺血管内皮细胞的作用；
甘露醇具有增加渗透压和清除自由基的作用；灌洗前肺动脉内注射PGE；
及增加 ECS渗透压和代谢底物、提高PH值使之略微偏碱均有助于抑制
供体肺离体、低温、冷缺血保存期间OFR的产生。
3．VV、SV、RSV作为肺泡上皮细胞核／质形态学变化的量化指标，体
视学定量原理在供肺保存质量研究中具有实用性。
OBJECTIVES
The purposes of this study were (I) to investigate improved Euro-Collins
solution (IECS) after analyzing the ingredient of Euro-Collins solution (ECS)
and modified Euro-Collins solution (MECS), (2) to study on the variation of
NO, MDA, SOD and the variety of morphology in isolated canine donor lung
followed by cold ischemic time, (3) to evaluate the applicability of quantized
principle of Biological Stereology in research of donor lung and to provide
experimental basis for observing in the model of the donor lung.
MATERALS AND METHODS
Twelve adult mongrel canines of male, weighing 19-24kg, anesthetized
with sodium pentobarbital, were randomly assigned to donor group (n=6) and
recipient group (n=6). The donor group was divided into control and
experiment group still further before and after perfusion. After the pulmonary
artery of donor injected by prostaglandin E1 (POEI, 4OO~.tg), the lung was
flushed by IECS (1 500ml) with direct single flush perfusion (SFP) at 4 0C
and pressure in the master of 3-4 kPa. Then to take out the whole of heart and
lungs, one of the lungs was for transplantation and the other stored for
determining the changes of molecule biochemistry and morphology
involving W/D and the concentrations of NO, SOD, MDA at various time.
The quantity stereological parameters of the nucleolus /cytoplasm volume
density (Vv) , surface density (Sv) and ratio of surface to volume for X phase
(Rsv) of the alveolar epithelia cells were measured and characterized by the
4
square net testing system of Biological Stereology. Recipient group: one side
of total pneumonectomy was performed, and then, the single lung
transplantation was finished at the same side. The circulation and respiration
functional parameters were monitored before, during and after
transplantation.
RESULTS
Comparison of control and experimental groups, no differences were found
in W/D, NO, SOD, MDA, bleeding grade in alveolus and Vv, Sv, Rsv of the
nucleolus /cytoplasm in the alveolar epithelia cells (P> 0.05), but there were
the downtrends in NO, SOD and Vv, Sv, and the uptrends in MDA, W/D and
Rsv along with prolonging time. The main changes were not haemorrhage in
tissues and alveolus of donor lung, but the pulmonary edema was existent in
there.
CONCLUSIONS
1. The characteristics of W/D, NO, SOD, MDA, bleeding grade in alveolus,
Vv, Sv and Rsv in the nucleolus /cytoplasm of the alveolar epithelia cells in
the isolated donor lung showed that the morphological changes were later
than OFR. The pulmonary edema was gradual evidence along with
prolonging time.
2. In this experimental conditions, the VEC had not damnified obviously
by perfusion with IECS in 4 hours. The pulmonary vascular resistance could
decreased by POE1 and so vascular endolhelial cells would be protected.
Mannitol not only increase osmotic pressure but also cleanse OFR. The
addition of mannitol, POE1, glucose and osmotic pressure to IEC solution
contributes to reduce the quantities of OFR.
3. The quantity stereological parameters of the nucleolus /cytoplasm Vv
and Sv and Rsv of the alveolar epithelia cells were the measure parameters of
alveolar epithelia cells. Biological Stereology may be applied to the
experimental study of lung preservation.
在分析ECS及MECS的基础上修改配方成分自制IECS；探讨犬肺
低温灌洗、离体保存过程中随着冷缺血时间推移NO、MDA、SOD的
变化规律；探讨分子水平肺组织活性改变与形态学变化的相互关系以及
体视学量化原理在供体肺研究中的可适用性。
材料和方法
12只雄性健康杂种犬随机分入供体组和受体组，供体组之供肺以
灌洗前为对照组；灌洗后低温保存期间不同时间点为实验观察组。麻醉
生效后，供体组：开胸后经肺动脉插管注入PGE_1400μg，采用低温单次
原位顺行法灌洗4℃ IECS 1500ml，压力控制在3～4kPa。灌洗完毕后
完整摘取心肺，一侧肺作为供肺移植，另一侧肺离体低温（4℃）保存，
测试、观察不同时间点肺组织分子生化和形态学改变，包括：肺组织
W/D；制备匀浆，测定肺组织内NO、MDA和SOD浓度；肺组织形态
学变化的定性观察及描述；用方网测试系统体视学量化原理测定肺泡上
皮细胞核/质Vv、Sv、Rsv。受体组：开胸后行单侧全肺切除并修整残
端，待移植肺残端修整后与之吻合；移植前、中、后监测血流动力学及
呼吸各项参数指标。
结 果
实验组与对照组相比较：W/D，NO、SOD和MDA含量，肺泡内
出血分级，肺泡上皮细胞核/质Vv、Sv、Rsv随低温保存时间变化无显
著性差异（P＞0.05）；但随时间延长NO、SOD、Vv、Sv均呈下降趋势，
而MDA、W／D、RSV却略有上升趋势。保存期间细小支气管上皮细胞
和血管内皮细胞结构正常，肺组织、肺泡内出血变化不明显，主要改变
是组织水肿。
结 论
1艰据犬肺离体低温保存后肺组织Wto，NO、SOD和 MDA含量，
肺泡内出血分级，肺泡上皮细胞核／质VV、SV、RSV随时间延长变化的
特点，可见肺组织病理形态学改变略滞后于OFR变化，随着保存时间
延长肺组织水肿渐趋明显。
2在本实验条件下所采用的IECS进行犬肺灌洗、离体保存4h之内
肺血管内皮细胞无明显损伤，PGE；具有保护肺血管内皮细胞的作用；
甘露醇具有增加渗透压和清除自由基的作用；灌洗前肺动脉内注射PGE；
及增加 ECS渗透压和代谢底物、提高PH值使之略微偏碱均有助于抑制
供体肺离体、低温、冷缺血保存期间OFR的产生。
3．VV、SV、RSV作为肺泡上皮细胞核／质形态学变化的量化指标，体
视学定量原理在供肺保存质量研究中具有实用性。
OBJECTIVES
The purposes of this study were (I) to investigate improved Euro-Collins
solution (IECS) after analyzing the ingredient of Euro-Collins solution (ECS)
and modified Euro-Collins solution (MECS), (2) to study on the variation of
NO, MDA, SOD and the variety of morphology in isolated canine donor lung
followed by cold ischemic time, (3) to evaluate the applicability of quantized
principle of Biological Stereology in research of donor lung and to provide
experimental basis for observing in the model of the donor lung.
MATERALS AND METHODS
Twelve adult mongrel canines of male, weighing 19-24kg, anesthetized
with sodium pentobarbital, were randomly assigned to donor group (n=6) and
recipient group (n=6). The donor group was divided into control and
experiment group still further before and after perfusion. After the pulmonary
artery of donor injected by prostaglandin E1 (POEI, 4OO~.tg), the lung was
flushed by IECS (1 500ml) with direct single flush perfusion (SFP) at 4 0C
and pressure in the master of 3-4 kPa. Then to take out the whole of heart and
lungs, one of the lungs was for transplantation and the other stored for
determining the changes of molecule biochemistry and morphology
involving W/D and the concentrations of NO, SOD, MDA at various time.
The quantity stereological parameters of the nucleolus /cytoplasm volume
density (Vv) , surface density (Sv) and ratio of surface to volume for X phase
(Rsv) of the alveolar epithelia cells were measured and characterized by the
4
square net testing system of Biological Stereology. Recipient group: one side
of total pneumonectomy was performed, and then, the single lung
transplantation was finished at the same side. The circulation and respiration
functional parameters were monitored before, during and after
transplantation.
RESULTS
Comparison of control and experimental groups, no differences were found
in W/D, NO, SOD, MDA, bleeding grade in alveolus and Vv, Sv, Rsv of the
nucleolus /cytoplasm in the alveolar epithelia cells (P> 0.05), but there were
the downtrends in NO, SOD and Vv, Sv, and the uptrends in MDA, W/D and
Rsv along with prolonging time. The main changes were not haemorrhage in
tissues and alveolus of donor lung, but the pulmonary edema was existent in
there.
CONCLUSIONS
1. The characteristics of W/D, NO, SOD, MDA, bleeding grade in alveolus,
Vv, Sv and Rsv in the nucleolus /cytoplasm of the alveolar epithelia cells in
the isolated donor lung showed that the morphological changes were later
than OFR. The pulmonary edema was gradual evidence along with
prolonging time.
2. In this experimental conditions, the VEC had not damnified obviously
by perfusion with IECS in 4 hours. The pulmonary vascular resistance could
decreased by POE1 and so vascular endolhelial cells would be protected.
Mannitol not only increase osmotic pressure but also cleanse OFR. The
addition of mannitol, POE1, glucose and osmotic pressure to IEC solution
contributes to reduce the quantities of OFR.
3. The quantity stereological parameters of the nucleolus /cytoplasm Vv
and Sv and Rsv of the alveolar epithelia cells were the measure parameters of
alveolar epithelia cells. Biological Stereology may be applied to the
experimental study of lung preservation.
引文
1． Muller C, Furst H, Reichenspurner H, et al. Lung procurement by low potassium dextran and the effect on preservation injury.Transplantation, 1999 ,68(8) : 1139-43
2． Roberts RF, Nishanian GP, Carey JN, et al. Addition of aprotinin to organ preservation solutions decreases lung reperfusion injury. Ann Thorac Surg ,1998,66(1) :225-30
3． Delima NF,Binns OA,Bochanan SA, et al.Low-potassium solution for lung preservation in the setting of high-flow reperfusion. Ann Thorac Surg, 1996 ,67(3) :973-6
4． Hopkinson DN, Bhabra MS, Hooper TL. Pulmonary graft preservation: a worrldwise survey of current clinical practice. J Heart Lung Transplant, 19 98,17(5) :525-8
5． 李刚，张临友，谭佩林等．低温灌洗UW液和Euro-Collins液保存离体鼠 肺效果的形态学比较研究．哈尔滨医科大学学报，2000，34(2) ：501-4
6． Cooper GD,Person FG,Patterson GA.Technique of successful lung transplantation in humans. J Thorac Cardiovasc Surg, 1987,93(2) : 173-81
7． Kon ND, Hines MH, Harr CD, et al. Improved lung preservation with cold air storage. Ann Thorac Surg,1991,51(4) :557-61
8． Wittwer T, Wahlers T, Elkis G, et al. Aprotinin enhanced lung preservation solutions: is it worthwhile. Ann Thorac Surg, 1999,67(5) : 1538-40
9． Guohu L, Shengxi C, Wanzhon L. Protective effects of ischemic preconditioning on donor lung in canine lung transplantation. Chest, 1998,113(5) : 1356-9
10． Shimokawa S, Matsunaga Y, Watanabe S, et al. A new lung preservation method of topical cooling by ambient cold air combined with high frequency scilation: An experimental study. Transplantation Proceedings, 1998 ,26(4) :2364-6
11． Struber M, Harringer W, Ernst M, et al. Inhaled nitric oxide as a prohyla-
ctic treatment against reperfusion injury of lung. J Thorac Cardiovasc Surg, 1999,47(3) : 179-82
12． 张志泰，陈玉平，宋飞强，等．单肺移植供肺采取及保护．中华器官移植杂 志，1997，18(4) ：242
13． Date H, Matsumura A, Manchester JK, et al. Changes in alveolar oxygen and carbon dioxide concentration and oxygen consumption during lung preservation. The maintenance of aerobic metabolism during lung preservation. J Thorac Cardiovasc Surg,1993,105(3) :492-501
14． Isselhard W, Minor T. Gaseous oxgen for protection and conditioning of organs during ischemia. Zentralbal Chir,1999,124(4) :252-9
15． Takigami K,Sasaki S,Shiiya N,et al.Evaluation of 18 hours lung preservation with oxygenated blood for optimal oxygen delivery.Ann Thorac Surg ,1997,66(2) :362-6
16． Puskas JD, Hirai T, Christie N, et al.Reliabe thirty-hour lung preservation by donor lung hyper-inflation.J Thorac Cardiovasc Surg, 1992,104(4) : 107 5
17． Shiraishi T,Igisu H,Shirakusa T.Effects of pH and temperature on lung preservation: a study with an isolated rat lung reperfusion model. Ann Thor ac Surg,1994,57(3) :639-43
18． Hiramatsu Y, Murao R,Chiba,Y, et al. Influence of pH of preservation solution on lung viability.Ann Thorac Surg,1994,58(4) :1083-6
19． Featherstone RL, Chambrs DJ, Kelly FJ, et al.Ischemical preconditioning enhance recovery of isolated rat lungs after hypothermic preservation. An-n Thorac Surg,2000,69(l):237-42
20． Novick RJ, Reid KR, Denning L, et al.Prelonged preservation of canine lung allografts:the role of postaglandins.Ann Thorac Surg,1991,51(5) :853-9
21 .Ueno T,Yolimise H,Oka T,et al. The effect of PGE, and temperature on lung function following preservation.Transplantation,1991,52(4) :626-30
22． Hooper TL,Fetherston GJ,Flecknell PA, et al.The use of a prostacyclin analog, iloprost, as an adjunct to pulmonary preservation with Euro-Collins solution. Transplantation,1990,49(3) :495-9
23． Chiang CH,Wu K,Yu CP, et al.Hypothermia and prostaglandin E1 produce synergistic at teuation of ischemia reperfusion lung injury. J Am Respir Crit Care Med, 1999,160(4) : 1319-23
24． Baker CJ,Longoria J,Gade PV, et al. Addition of a water soluble alph-toco-pherol analogue to UW solution improves endothelial viability and decreases lung reperfusion injury. J Surg Res, 1999,86(1) : 145-9
25． FukuseT,Jojn H,Tan W,et al.Influence of deflated and anaerobic condition during cold storage on rat lungs. Am J Respir Crit Care Med, 1999,160(2) :621-7
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33． Novick RJ,Gehman KE, Ali IS, et al. Lung preservation:the importance of endothelial and alveolar type Ⅱ cell integrity. Ann Thorac Surg, 1996, 62 (1) :302-14
34． Haniuda M,Dresler CM,Mizuta T,et al. Free radical mediated vascular in-
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2． Roberts RF, Nishanian GP, Carey JN, et al. Addition of aprotinin to organ preservation solutions decreases lung reperfusion injury. Ann Thorac Surg ,1998,66(1) :225-30
3． Delima NF,Binns OA,Bochanan SA, et al.Low-potassium solution for lung preservation in the setting of high-flow reperfusion. Ann Thorac Surg, 1996 ,67(3) :973-6
4． Hopkinson DN, Bhabra MS, Hooper TL. Pulmonary graft preservation: a worrldwise survey of current clinical practice. J Heart Lung Transplant, 19 98,17(5) :525-8
5． 李刚，张临友，谭佩林等．低温灌洗UW液和Euro-Collins液保存离体鼠 肺效果的形态学比较研究．哈尔滨医科大学学报，2000，34(2) ：501-4
6． Cooper GD,Person FG,Patterson GA.Technique of successful lung transplantation in humans. J Thorac Cardiovasc Surg, 1987,93(2) : 173-81
7． Kon ND, Hines MH, Harr CD, et al. Improved lung preservation with cold air storage. Ann Thorac Surg,1991,51(4) :557-61
8． Wittwer T, Wahlers T, Elkis G, et al. Aprotinin enhanced lung preservation solutions: is it worthwhile. Ann Thorac Surg, 1999,67(5) : 1538-40
9． Guohu L, Shengxi C, Wanzhon L. Protective effects of ischemic preconditioning on donor lung in canine lung transplantation. Chest, 1998,113(5) : 1356-9
10． Shimokawa S, Matsunaga Y, Watanabe S, et al. A new lung preservation method of topical cooling by ambient cold air combined with high frequency scilation: An experimental study. Transplantation Proceedings, 1998 ,26(4) :2364-6
11． Struber M, Harringer W, Ernst M, et al. Inhaled nitric oxide as a prohyla-
ctic treatment against reperfusion injury of lung. J Thorac Cardiovasc Surg, 1999,47(3) : 179-82
12． 张志泰，陈玉平，宋飞强，等．单肺移植供肺采取及保护．中华器官移植杂 志，1997，18(4) ：242
13． Date H, Matsumura A, Manchester JK, et al. Changes in alveolar oxygen and carbon dioxide concentration and oxygen consumption during lung preservation. The maintenance of aerobic metabolism during lung preservation. J Thorac Cardiovasc Surg,1993,105(3) :492-501
14． Isselhard W, Minor T. Gaseous oxgen for protection and conditioning of organs during ischemia. Zentralbal Chir,1999,124(4) :252-9
15． Takigami K,Sasaki S,Shiiya N,et al.Evaluation of 18 hours lung preservation with oxygenated blood for optimal oxygen delivery.Ann Thorac Surg ,1997,66(2) :362-6
16． Puskas JD, Hirai T, Christie N, et al.Reliabe thirty-hour lung preservation by donor lung hyper-inflation.J Thorac Cardiovasc Surg, 1992,104(4) : 107 5
17． Shiraishi T,Igisu H,Shirakusa T.Effects of pH and temperature on lung preservation: a study with an isolated rat lung reperfusion model. Ann Thor ac Surg,1994,57(3) :639-43
18． Hiramatsu Y, Murao R,Chiba,Y, et al. Influence of pH of preservation solution on lung viability.Ann Thorac Surg,1994,58(4) :1083-6
19． Featherstone RL, Chambrs DJ, Kelly FJ, et al.Ischemical preconditioning enhance recovery of isolated rat lungs after hypothermic preservation. An-n Thorac Surg,2000,69(l):237-42
20． Novick RJ, Reid KR, Denning L, et al.Prelonged preservation of canine lung allografts:the role of postaglandins.Ann Thorac Surg,1991,51(5) :853-9
21 .Ueno T,Yolimise H,Oka T,et al. The effect of PGE, and temperature on lung function following preservation.Transplantation,1991,52(4) :626-30
22． Hooper TL,Fetherston GJ,Flecknell PA, et al.The use of a prostacyclin analog, iloprost, as an adjunct to pulmonary preservation with Euro-Collins solution. Transplantation,1990,49(3) :495-9
23． Chiang CH,Wu K,Yu CP, et al.Hypothermia and prostaglandin E1 produce synergistic at teuation of ischemia reperfusion lung injury. J Am Respir Crit Care Med, 1999,160(4) : 1319-23
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