左旋精氨酸对同期单侧移植肺及离体肺保护作用的研究
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摘要
肺移植已经成为一种治疗终末期肺疾病的有效手段。但其发病率及死亡率明显高于心脏、肝脏及肾脏移植。其中缺血再灌注损伤对移植肺功能的影响是引起移植后肺功能障碍和受体死亡的主要原因。因此，对肺缺血再灌注损伤的保护是提高肺移植成功率的一个重要措施。此外，由于应用现有供肺保存技术所能提供的肺安全缺血时间的限制，使得供肺仍然十分短缺，改进并完善供肺保存技术显得尤为重要。
内皮细胞所释放的一氧化氮(Nitric oxide，NO)在保持肺循环稳态中起着非常重要的作用，NO可调节肺血管张力、保护肺毛细血管膜的完整性、抑制血小板聚集和白细胞粘附的作用。在肺移植过程中NO明显减少。研究己证实，给予NO或NO合成供体能明显改善移植心脏或肝脏的功能。
本实验在犬单肺移植模型的基础上，采用加入NO合成前体左旋精氨酸(L-Arginine，L-Arg)的冷Euro-Collins(EC)液灌注并离体低温保存犬肺，观察了L-Arg对离体供肺和肺移植缺血再灌注损伤的保护作用，并对其可能机制进行了探讨。
1 L-Arg在供肺离体保存中的作用
1．1 目的：通过采用犬单肺移植模型，探讨L-Arg在供肺离体保存中的作用及其可能机制。
1．2 方法：将实验犬10只，随机分为2组，对照组以4℃EC液
第四军医大学硕士学位论文
灌注及保存供肺，实验组以4℃含L一Arg(2留L)的EC液灌注及
保存供肺。分别于肺离体低温保存30、60、120、180、240min
后取材，测定不同时间点NO、超氧化物歧化酶(SOD)和丙二
醛(MDA)含量变化及240min肺组织的湿干重量t匕(W/D)和
组织结构的改变，以评价L一Aig对供肺的保护效果。
L3结果:两组在不同时间点NO、SOD、MDA的含量和W心
有显著性差异(尸<0.05，尸< 0.01);实验组肺组织中NO及SOD
含量较对照组明显增高，MDA含量显著降低。随时间延长NO，
SOD均有下降趋势，但实验组下降幅度较对照组小。MDA均轻
度上升，但实验组上升幅度较对照组小。W月〕和光镜检查发现，
实验组损伤明显轻于对照组(尸<0.05)。
1.4结论:在EC液中加入L一Arg用于离体肺的灌注和保存，能
明显减轻供肺损伤，其机制可能与NO生成增加、氧自由基生成
及清除过程的变化有关。
ZL一Arg对肺移植缺血再灌注损伤的保护作用
2.1目的:通过采用犬单肺移植模型，观察L一赶g对肺移植缺血
再灌注损伤的保护作用及其机制。
2.2方法:实验犬16只，随机分为2组，每组8只。肺灌注液及
保存液均使用EC液，其中对照组仅用4℃的EC液对供肺进行
灌洗和保存;实验组用4℃含L一Arg(Zg/L)的Ec液对供肺进行
灌洗和保存，然后进行左肺移植。于移植肺再灌注后lh取肺组
织，测定不同时间点NO、髓过氧化物酶(MPO)活性;在肺
移植前及移植再灌注后lh于股静脉采血，测定血浆内皮素
(ET一1)、血栓素B:(TXBZ)、前列腺素F，。(6一Keto一PGF!。)
含量。实验结束前，测定移植肺含水量。
2.3结果:再灌注后lh，实验组肺组织中NO含量明显高于对照
组(尸<0.01);MPO活性在实验组则明显低于对照组(P<0.01)。
第四军医大学硕士学位论文
血浆中ET一1含量对照组明显高于实验组(P<0.05)。肺动脉开放后
lh，两组动物血浆TXB:水平均显著升高，但对照组表现的更加
明显，组间差异显著(尸<0.01)。而6一Keto一PGFla水平则在肺移
植前后及两组之间无明显差异。对照组W心值明显高于实验组
(P<0 .05)。
2.4结论:在犬肺移植过程中，在保存及灌洗液中加入L一Arg可
使NO含量增加，减少ET一1的产生、减轻中性粒细胞的聚集、
调节PGIZ与TXA:的相对平衡，从而加强对肺的保护作用，减轻
肺缺血再灌注损伤。
通过上述结论，本研究表明在保存及灌洗液中加入外源性NO供
体L一Arg可加强对犬肺的保护作用，减轻肺缺血再灌注损伤，改
善移植肺的功能。
Lung transplantation is actually considered as an effective therapy in end-staged pulmonary disease. Compared with the heart, liver, or kidney transplantation, its morbidity and mortality remained high, however. Ischemia-reperfusion-induced lung injury has been identified as the main cause of primary graft failure and mortality after lung transplantation, its prevention and treatment can be anticipated with much more success. In addition, the donor lung is still rather scanty because the ischemic time provided by preservation technique available at present is limited. It is therefore necessary to explore and improve the technique of donor lung preservation.
Constitutive expression of endogenous nitric oxide (NO) by the pulmonary vascular endothelium plays an important role in maintaining the homeostasis in many aspects of the pulmonary circulation, including modulation of pulmonary vascular tone, maintenance of an intact pulmonary capillary membrane, inhibition of platelet aggregation and leukocyte adhesion. A marked decrease of endogenous NO has been found during lung transplantation. Previous studies have demonstrated that administration of NO or NO donors may greatly improve the function of heart and liver after transplantation.
We used a canine single lung transplantation model to evaluate the protective benefits of L-Arginine (L-Arg), a substrate of endogenous nitric oxide synthesis, to the isolated donor lung or to
ischemia-reperfusion-induced injury lung by adding L-Arg as an additive to the cooling preservation and flush EC solution. Moreover, we tried to explore the possible mechanisms.
Part 1 Effect of L-Arg on isolated donor lung preservation
1.1 Objective: To investigate the protective effect of L-Arg on the preservation of isolated donor lung and the possible mechanisms based on a canine single lung transplantation model.
1.2 Methods: Ten dogs were randomly divided into 2 groups. In the control group, the isolated donor lungs were flushed with and preserved in cold Euro-collins solution(EC) at 4 C . In the experimental group, the isolated donor lungs were flushed with and preserved in cold EC added with 2g/L L-Arg at 4 C. After preservation for 30, 60, 120, 180, 240min respectively, the tissue samples of donor lung were subsequently obtained for the measurement of nitric oxide (NO), superoxide dismutase (SOD) and malondialdehyde (MDA) contents. In addition, the wet to dry weight ratio (W/D) and histological changes were also observed at 240min.
1.3 Results: The comparison of control and experimental groups revealed that there were significant differences in NO, SOD, MDA contents and W/D at any appointed time points (P<0.05, P<0.01). The NO and SOD contents of experimental groups were significantly higher than that of the control group. But the MDA contents were lower in the experimental group. Furthermore, The NO and SOD contents tended to drop while MDA level rose as the duration of preservation was lengthened. These phenomena were more prominent in the control group. The W/D and light microscope revealed that the control group was more seriously damaged (P<0.05).
1.4 Conclusion: L-Arg added in EC has a significant protective effect in isolated donor lung preservation. The increased NO production and changes of the balance between clearout and
production of oxygen-free radicals are likely the main mechanisms.
Part 2 Effect of L-Arg on ischemia-reperfusion injury in lung transplantation
2.1 Objective: To investigate the protective effect of L-Arg on ischemia-reperfusion injury in lung transplantation and possible mechanisms based on a canine single lung transplantation model.
2.2 Methods: Sixteen dogs were randomly divided into 2 groups. In the control group, the donor lungs were flushed with and preserved in the cold EC at 4C. In the experimental group, the donor lungs were flushed with and preserved in cold EC added with 2g/L L-Arg at 4C. After 4 h preservation, the left lungs were implanted into
内皮细胞所释放的一氧化氮(Nitric oxide，NO)在保持肺循环稳态中起着非常重要的作用，NO可调节肺血管张力、保护肺毛细血管膜的完整性、抑制血小板聚集和白细胞粘附的作用。在肺移植过程中NO明显减少。研究己证实，给予NO或NO合成供体能明显改善移植心脏或肝脏的功能。
本实验在犬单肺移植模型的基础上，采用加入NO合成前体左旋精氨酸(L-Arginine，L-Arg)的冷Euro-Collins(EC)液灌注并离体低温保存犬肺，观察了L-Arg对离体供肺和肺移植缺血再灌注损伤的保护作用，并对其可能机制进行了探讨。
1 L-Arg在供肺离体保存中的作用
1．1 目的：通过采用犬单肺移植模型，探讨L-Arg在供肺离体保存中的作用及其可能机制。
1．2 方法：将实验犬10只，随机分为2组，对照组以4℃EC液
第四军医大学硕士学位论文
灌注及保存供肺，实验组以4℃含L一Arg(2留L)的EC液灌注及
保存供肺。分别于肺离体低温保存30、60、120、180、240min
后取材，测定不同时间点NO、超氧化物歧化酶(SOD)和丙二
醛(MDA)含量变化及240min肺组织的湿干重量t匕(W/D)和
组织结构的改变，以评价L一Aig对供肺的保护效果。
L3结果:两组在不同时间点NO、SOD、MDA的含量和W心
有显著性差异(尸<0.05，尸< 0.01);实验组肺组织中NO及SOD
含量较对照组明显增高，MDA含量显著降低。随时间延长NO，
SOD均有下降趋势，但实验组下降幅度较对照组小。MDA均轻
度上升，但实验组上升幅度较对照组小。W月〕和光镜检查发现，
实验组损伤明显轻于对照组(尸<0.05)。
1.4结论:在EC液中加入L一Arg用于离体肺的灌注和保存，能
明显减轻供肺损伤，其机制可能与NO生成增加、氧自由基生成
及清除过程的变化有关。
ZL一Arg对肺移植缺血再灌注损伤的保护作用
2.1目的:通过采用犬单肺移植模型，观察L一赶g对肺移植缺血
再灌注损伤的保护作用及其机制。
2.2方法:实验犬16只，随机分为2组，每组8只。肺灌注液及
保存液均使用EC液，其中对照组仅用4℃的EC液对供肺进行
灌洗和保存;实验组用4℃含L一Arg(Zg/L)的Ec液对供肺进行
灌洗和保存，然后进行左肺移植。于移植肺再灌注后lh取肺组
织，测定不同时间点NO、髓过氧化物酶(MPO)活性;在肺
移植前及移植再灌注后lh于股静脉采血，测定血浆内皮素
(ET一1)、血栓素B:(TXBZ)、前列腺素F，。(6一Keto一PGF!。)
含量。实验结束前，测定移植肺含水量。
2.3结果:再灌注后lh，实验组肺组织中NO含量明显高于对照
组(尸<0.01);MPO活性在实验组则明显低于对照组(P<0.01)。
第四军医大学硕士学位论文
血浆中ET一1含量对照组明显高于实验组(P<0.05)。肺动脉开放后
lh，两组动物血浆TXB:水平均显著升高，但对照组表现的更加
明显，组间差异显著(尸<0.01)。而6一Keto一PGFla水平则在肺移
植前后及两组之间无明显差异。对照组W心值明显高于实验组
(P<0 .05)。
2.4结论:在犬肺移植过程中，在保存及灌洗液中加入L一Arg可
使NO含量增加，减少ET一1的产生、减轻中性粒细胞的聚集、
调节PGIZ与TXA:的相对平衡，从而加强对肺的保护作用，减轻
肺缺血再灌注损伤。
通过上述结论，本研究表明在保存及灌洗液中加入外源性NO供
体L一Arg可加强对犬肺的保护作用，减轻肺缺血再灌注损伤，改
善移植肺的功能。
Lung transplantation is actually considered as an effective therapy in end-staged pulmonary disease. Compared with the heart, liver, or kidney transplantation, its morbidity and mortality remained high, however. Ischemia-reperfusion-induced lung injury has been identified as the main cause of primary graft failure and mortality after lung transplantation, its prevention and treatment can be anticipated with much more success. In addition, the donor lung is still rather scanty because the ischemic time provided by preservation technique available at present is limited. It is therefore necessary to explore and improve the technique of donor lung preservation.
Constitutive expression of endogenous nitric oxide (NO) by the pulmonary vascular endothelium plays an important role in maintaining the homeostasis in many aspects of the pulmonary circulation, including modulation of pulmonary vascular tone, maintenance of an intact pulmonary capillary membrane, inhibition of platelet aggregation and leukocyte adhesion. A marked decrease of endogenous NO has been found during lung transplantation. Previous studies have demonstrated that administration of NO or NO donors may greatly improve the function of heart and liver after transplantation.
We used a canine single lung transplantation model to evaluate the protective benefits of L-Arginine (L-Arg), a substrate of endogenous nitric oxide synthesis, to the isolated donor lung or to
ischemia-reperfusion-induced injury lung by adding L-Arg as an additive to the cooling preservation and flush EC solution. Moreover, we tried to explore the possible mechanisms.
Part 1 Effect of L-Arg on isolated donor lung preservation
1.1 Objective: To investigate the protective effect of L-Arg on the preservation of isolated donor lung and the possible mechanisms based on a canine single lung transplantation model.
1.2 Methods: Ten dogs were randomly divided into 2 groups. In the control group, the isolated donor lungs were flushed with and preserved in cold Euro-collins solution(EC) at 4 C . In the experimental group, the isolated donor lungs were flushed with and preserved in cold EC added with 2g/L L-Arg at 4 C. After preservation for 30, 60, 120, 180, 240min respectively, the tissue samples of donor lung were subsequently obtained for the measurement of nitric oxide (NO), superoxide dismutase (SOD) and malondialdehyde (MDA) contents. In addition, the wet to dry weight ratio (W/D) and histological changes were also observed at 240min.
1.3 Results: The comparison of control and experimental groups revealed that there were significant differences in NO, SOD, MDA contents and W/D at any appointed time points (P<0.05, P<0.01). The NO and SOD contents of experimental groups were significantly higher than that of the control group. But the MDA contents were lower in the experimental group. Furthermore, The NO and SOD contents tended to drop while MDA level rose as the duration of preservation was lengthened. These phenomena were more prominent in the control group. The W/D and light microscope revealed that the control group was more seriously damaged (P<0.05).
1.4 Conclusion: L-Arg added in EC has a significant protective effect in isolated donor lung preservation. The increased NO production and changes of the balance between clearout and
production of oxygen-free radicals are likely the main mechanisms.
Part 2 Effect of L-Arg on ischemia-reperfusion injury in lung transplantation
2.1 Objective: To investigate the protective effect of L-Arg on ischemia-reperfusion injury in lung transplantation and possible mechanisms based on a canine single lung transplantation model.
2.2 Methods: Sixteen dogs were randomly divided into 2 groups. In the control group, the donor lungs were flushed with and preserved in the cold EC at 4C. In the experimental group, the donor lungs were flushed with and preserved in cold EC added with 2g/L L-Arg at 4C. After 4 h preservation, the left lungs were implanted into
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