缺血后处理对大鼠无心跳供体肺移植的保护作用
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摘要
目的：
建立大鼠无心跳供体肺移植动物模型，观察尸体肺不同早期保护措施的保护效果；研究缺血后处理对肺移植缺血再灌注损伤的保护作用并初步探讨其保护机制。
材料与方法：
第一部分：64只大鼠随机分成4组，即有心跳供体组(HBD组)，无心跳供体-供肺未采取保护措施组(NHBD-N组)，无心跳供体-供肺持续机械通气组(NHBD-V组)和无心跳供体-供肺冰盐水浸泡组(NHBD-I组)；第二部分：40只大鼠随机分成2组，对照组(NHBD-C组)，即NHBD肺移植后直接开放肺动脉，和缺血后处理组(NHBD-S组)，即NHBD肺移植后肺动脉给予1min再灌-1min再阻断，反复5次的后处理措施，然后全面恢复再灌注。
观察指标：
术后短期存活率：观察各组受体大白鼠左肺原位移植术后短期存活率，以术后2h心脏维持跳动为标准；在结扎右肺门前和结扎后15、30 min3个时间段测定肺顺应性、抽颈动脉血查血气分析并计算氧合指数；在结扎右肺门后1h或受体大白鼠死亡时，取移植的左肺肺组织测定肺水含量、肺组织能量代谢物含量和形态学观察；另外，第二部分在结扎右肺门后1h或受体大白鼠死亡时，取移植的左肺肺组织用TUNEL法行细胞凋亡检测、用RT-PCR测定移植肺ICAM-1 mRNA的表达和SOD、MDA含量的测定，并在电镜下观察移植肺组织超微结构的改变。
统计方法：
所有数据以均数±标准差((?)±s)表示，用SPSS12.0统计软件自动分析，比较两组之间的差异采用student t检验，多组间的差异比较采用单因数方差分析；统计结果处理：以P＜0.05表示差异有统计意义；P＜0.01表示差异有高度统计意义。
结果：
1．术后短期存活率：第一部分和第二部分各组术后短期存活率均很高，组间无明显差异，p＞0.05。
2．肺顺应性：在第一部分，NHBD-N组与HBD组比较移植后肺顺应性较差(P＜0.01)，而NHBD-V和NHBD-I组与HBD组比较无显著差异；在第二部分，NHBD-S组移植术后肺顺应性较NHBD-C组要高，两组比较在结扎右肺门前(P＜0.05)和结扎后15min(P＜0.01)差异具有显著性。
3．动脉血气分析：在第一部分，实验结果显示各组之间肺功能指标相差不大，NHBD-N组在结扎右肺门后30 min与HBD组比较PaO2较低，有显著差异(P＜0.05)。其余比较未见显著差异；在第二部分，NHBD-S组左肺移植术后鼠肺功能指标较NHBD-C好(P＜0.05)。
4．肺水含量：在第一部分，NHBD-N与其它各组比较肺湿／干重比值高，差异具有显著性(P＜0.01)。其它3组间无差异性；在第二部分，实验结果显示NHBD-S组左肺移植术后鼠肺湿／干重比值明显较NHBD-C低，两者差异有高度统计意义(P＜0.01)。
5．肺组织中能量代谢物的含量：实验结果显示与HBD比较，其它各组肺能量代谢物都有所减低，但NHBD-I组相对肺能量代谢物的保存较NHBD-N和NHBD-V组要好，而NHBD-N组最差(P＜0.01)。
6．光镜检查：在第一部分，实验结果显示NHBD-N和NHBD-V组与HBD组比较，炎性细胞浸润程度和肺损伤程度较重，而NHBD-I组与HBD组较接近；在第二部分，两组肺移植术后均出现肺泡水肿，炎性细胞渗出，毛细血管破裂出血，肺间质水肿等等病理改变，但NHBD-S组损伤较轻。
7．移植肺细胞凋亡检测：NHBD-S组细胞凋亡的程度较NHBD-C组明显减轻，两者之间具有高度差异性(P＜0.01)。
8．肺组织SOD、MDA含量的检测：NHBD-S组MDA含量较NHBD-C低，而SOD含量较NHBD-C组高，差异具有高度统计学意义(P＜0.01)。
9．移植肺ICAM-1 mRNA表达：实验结果显示NHBD-C组移植术后肺组织ICAM-1 mRNA含量高于NHBD-S组(P＜0.01)。
10．电镜超微结构的改变：NHBD-S组肺超微结构的病变损伤较NHBD-C组轻。
结论：
1．用“两套管一支架管”法建立大鼠NHBD左肺原位移植模型是一种简单有效的方法，该模型适用于肺移植早期相关问题的研究。
2．热缺血30 min大白鼠无心跳供体肺适于肺移植。在建模过程中，供、受体大白鼠无需常规全身肝素化。
3．大鼠NHBD肺在热缺血时给予持续机械通气或胸腔低温浸泡都可取得良好的肺保护效果，而胸腔低温浸泡的肺保护效果要稍优于持续机械通气。
4．缺血后处理对无心跳供体肺移植具有保护作用，可减轻肺移植术后缺血再灌注损伤，减轻肺水肿、改善肺顺应性和保存移植肺肺功能和结构。
5．缺血后处理肺保护机制包括：抑制肺移植术后脂质过氧化反应减轻肺水肿；下调ICAM-1 mRNA的表达水平，抑制炎症反应和中性粒细胞活化；抑制肺移植术后细胞凋亡等。
创新和意义：
1．本研究在建立大鼠NHBD左肺原位移植模型的基础上，首次研究并比较尸体肺不同早期保护措施的效果。
2．本研究在实验过程中证实热缺血30 min大白鼠无心跳供体肺适于肺移植，并提出在建模过程中，供、受体大白鼠无需常规全身肝素化。
3．本研究国内外首次研究缺血后处理对NHBD肺移植缺血再灌注损伤的保护作用，并初步探讨其保护机制。
4．在研究其保护机制中发现，缺血后处理能抑制肺移植术后脂质过氧化反应、下调肺移植后肺组织ICAM-1mRNA表达和抑制肺移植术后细胞凋亡。
Objective:
To establish animal model of non-heart-beating donor in rat lungtransplantation and to observe the protective effect of various protectivemeasures on cadaver lung; to study the protective effect and mechanismof ischemic postconditioning on ischemic reperfusion injury in rat NHBDlung transplantation.
Methods:
PartⅠ: sixty four Sprague-Dawley rats were randomly divided intofour groups, heart-beating donor group (HBD group), non-heart-beatingdonor group without protective measure on cadaver lung (NHBD-Ngroup), non-heart-beating donor group with continuous mechanicalventilation on cadaver lung (NHBD-V group), and non-heart-beatingdonor group with topical cooling on cadaver lung by ice saline (NHBD-Igroup); PartⅡ: forty Sprague-Dawley rats were randomly divided intofour groups, namely control group (NHBD-C group) and ischemicpostconditioning group (NHBD-S group) with 5 cycles of 1 min'reperfusion and 1 min' reocclusion. Surviving rate in the observationperiod of 2 hour after transplantation, lung compliance, blood gas check, the wet/dry ratio of lung after transplantation, and pathological changeswere analyzed and compared among groups in PartⅠand PartⅡ.Besides, in PartⅠ, the amount of ATP、ADP and AMP in lung tissueafter transplantation were checked by high-performance liquidchromatography(HPLC), and the sum of adenylic nucleotides werecomputerized and compared among each group; in PartⅡ, the amount ofSOD and MDA, the expression of cell apoptosis and ICAM-1mRNA inlung tissue after transplantation were assessed, and ultramicroscopicstructural changes also observed in PartⅡ.
Results: (in PartⅠand PartⅡ)
1. Surviving rate: in PartⅠand PartⅡ, each group had highsurviving rate and there were no difference between each group.
2. Lung compliance: in PartⅠ, the lung compliance in NHBD-Ngroup was much worse than that in HBD group, and there wereno significant difference among HBD group、NHBD-V groupand NHBD-I group; in PartⅡ, the lung compliance in NHBD-Sgroup was much better that in control group.
3. Blood gas check: in PartⅠ, there were no significant differencebetween each group; in PartⅡ, the PaO2 and OI were higher inNHBD-S group compared with NHBD-C group.
4. The wet/dry ratio of lung: in PartⅠ, the wet/dry ratio of lung inNHBD-N group was much higher than that in other groups, there were no significant difference among HBD group、NHBD-Vgroup and NHBD-I group; in PartⅡ, the wet/dry ratio of lung inNHBD-S group was much lower than that in NHBD-C group (p＜0.01).
5. The amount of ATP、ADP and AMP: in PartⅠ, the amount ofATP、ADP and AMP and TAN in HBD group＞that in NHBD-Igroup＞that in NHBD-V group＞that in NHBD-N group.
6. Pathological changes: in PartⅠ, the lung histologicalexamination showed that pathological changes in NHBD-Ngroup and in NHBD-V group were more serious than that inHBD group, and the pathological changes in NHBD-I group wasclose to that in HBD group; in PartⅡ, the pathological changesin NHBD-C group were more serious than that in NHBD-Sgroup.
7. The expression of cell apoptosis: in PartⅡ, the expression of cellapoptosis in NHBD-C group were much higher than that inNHBD-S group.
8. The amount of SOD and MDA: in PartⅡ, the amount of SOD inlung tissue after transplantation in NHBD-S group was muchhigher than that in NHBD-C group; the amount of MDA in lungtissue after transplantation in NHBD-S group was much lowerthan that in NHBD-C group.
9. The expression of ICAM-1mRNA: assessed by RT-PCR, theexpression of ICAM-1mRNA in lung tissue after transplantationin NHBD-S group was obviously down-regulated compared withNHBD-C group.
10.Ultramicroscopic structural changes: Under electronicmicroscope, the ultramicroscopic structural changes in NHBD-Cgroup was more serious than that in NHBD-S group.
Conclusion:
1. Establishing animal model of non-heart-beating donor in rat lungtransplantation with two-cuff-one-stent technique is a simple andeffective method, which is fit for studying early problems inNHBD lung transplantation.
2. The NHBD lung with 30 min of WIT is suitable for lungtransplantation in rats; and routine heparinization in donor andreceptor is not necessary.
3. Excellent protective effect on cadaver lung can be reached bycontinuous ventilation or topical cooling, and the latter is a litterbetter than the former.
4. Ischemic postconditioning has protective effect on NHBD lungtransplantation, it may decrease ischemic reperfusion injury afterlung transplantation and preserve the structure and function oflung.
5. The mechanism of ischemic postconditioning includes: increasethe amount of SOD and decrease the amount of MDA; inhibit theexpression of cell apoptosis after transplantation; down-regulatethe expression of ICAM-1mRNA of lung tissue aftertransplantation.
建立大鼠无心跳供体肺移植动物模型，观察尸体肺不同早期保护措施的保护效果；研究缺血后处理对肺移植缺血再灌注损伤的保护作用并初步探讨其保护机制。
材料与方法：
第一部分：64只大鼠随机分成4组，即有心跳供体组(HBD组)，无心跳供体-供肺未采取保护措施组(NHBD-N组)，无心跳供体-供肺持续机械通气组(NHBD-V组)和无心跳供体-供肺冰盐水浸泡组(NHBD-I组)；第二部分：40只大鼠随机分成2组，对照组(NHBD-C组)，即NHBD肺移植后直接开放肺动脉，和缺血后处理组(NHBD-S组)，即NHBD肺移植后肺动脉给予1min再灌-1min再阻断，反复5次的后处理措施，然后全面恢复再灌注。
观察指标：
术后短期存活率：观察各组受体大白鼠左肺原位移植术后短期存活率，以术后2h心脏维持跳动为标准；在结扎右肺门前和结扎后15、30 min3个时间段测定肺顺应性、抽颈动脉血查血气分析并计算氧合指数；在结扎右肺门后1h或受体大白鼠死亡时，取移植的左肺肺组织测定肺水含量、肺组织能量代谢物含量和形态学观察；另外，第二部分在结扎右肺门后1h或受体大白鼠死亡时，取移植的左肺肺组织用TUNEL法行细胞凋亡检测、用RT-PCR测定移植肺ICAM-1 mRNA的表达和SOD、MDA含量的测定，并在电镜下观察移植肺组织超微结构的改变。
统计方法：
所有数据以均数±标准差((?)±s)表示，用SPSS12.0统计软件自动分析，比较两组之间的差异采用student t检验，多组间的差异比较采用单因数方差分析；统计结果处理：以P＜0.05表示差异有统计意义；P＜0.01表示差异有高度统计意义。
结果：
1．术后短期存活率：第一部分和第二部分各组术后短期存活率均很高，组间无明显差异，p＞0.05。
2．肺顺应性：在第一部分，NHBD-N组与HBD组比较移植后肺顺应性较差(P＜0.01)，而NHBD-V和NHBD-I组与HBD组比较无显著差异；在第二部分，NHBD-S组移植术后肺顺应性较NHBD-C组要高，两组比较在结扎右肺门前(P＜0.05)和结扎后15min(P＜0.01)差异具有显著性。
3．动脉血气分析：在第一部分，实验结果显示各组之间肺功能指标相差不大，NHBD-N组在结扎右肺门后30 min与HBD组比较PaO2较低，有显著差异(P＜0.05)。其余比较未见显著差异；在第二部分，NHBD-S组左肺移植术后鼠肺功能指标较NHBD-C好(P＜0.05)。
4．肺水含量：在第一部分，NHBD-N与其它各组比较肺湿／干重比值高，差异具有显著性(P＜0.01)。其它3组间无差异性；在第二部分，实验结果显示NHBD-S组左肺移植术后鼠肺湿／干重比值明显较NHBD-C低，两者差异有高度统计意义(P＜0.01)。
5．肺组织中能量代谢物的含量：实验结果显示与HBD比较，其它各组肺能量代谢物都有所减低，但NHBD-I组相对肺能量代谢物的保存较NHBD-N和NHBD-V组要好，而NHBD-N组最差(P＜0.01)。
6．光镜检查：在第一部分，实验结果显示NHBD-N和NHBD-V组与HBD组比较，炎性细胞浸润程度和肺损伤程度较重，而NHBD-I组与HBD组较接近；在第二部分，两组肺移植术后均出现肺泡水肿，炎性细胞渗出，毛细血管破裂出血，肺间质水肿等等病理改变，但NHBD-S组损伤较轻。
7．移植肺细胞凋亡检测：NHBD-S组细胞凋亡的程度较NHBD-C组明显减轻，两者之间具有高度差异性(P＜0.01)。
8．肺组织SOD、MDA含量的检测：NHBD-S组MDA含量较NHBD-C低，而SOD含量较NHBD-C组高，差异具有高度统计学意义(P＜0.01)。
9．移植肺ICAM-1 mRNA表达：实验结果显示NHBD-C组移植术后肺组织ICAM-1 mRNA含量高于NHBD-S组(P＜0.01)。
10．电镜超微结构的改变：NHBD-S组肺超微结构的病变损伤较NHBD-C组轻。
结论：
1．用“两套管一支架管”法建立大鼠NHBD左肺原位移植模型是一种简单有效的方法，该模型适用于肺移植早期相关问题的研究。
2．热缺血30 min大白鼠无心跳供体肺适于肺移植。在建模过程中，供、受体大白鼠无需常规全身肝素化。
3．大鼠NHBD肺在热缺血时给予持续机械通气或胸腔低温浸泡都可取得良好的肺保护效果，而胸腔低温浸泡的肺保护效果要稍优于持续机械通气。
4．缺血后处理对无心跳供体肺移植具有保护作用，可减轻肺移植术后缺血再灌注损伤，减轻肺水肿、改善肺顺应性和保存移植肺肺功能和结构。
5．缺血后处理肺保护机制包括：抑制肺移植术后脂质过氧化反应减轻肺水肿；下调ICAM-1 mRNA的表达水平，抑制炎症反应和中性粒细胞活化；抑制肺移植术后细胞凋亡等。
创新和意义：
1．本研究在建立大鼠NHBD左肺原位移植模型的基础上，首次研究并比较尸体肺不同早期保护措施的效果。
2．本研究在实验过程中证实热缺血30 min大白鼠无心跳供体肺适于肺移植，并提出在建模过程中，供、受体大白鼠无需常规全身肝素化。
3．本研究国内外首次研究缺血后处理对NHBD肺移植缺血再灌注损伤的保护作用，并初步探讨其保护机制。
4．在研究其保护机制中发现，缺血后处理能抑制肺移植术后脂质过氧化反应、下调肺移植后肺组织ICAM-1mRNA表达和抑制肺移植术后细胞凋亡。
Objective:
To establish animal model of non-heart-beating donor in rat lungtransplantation and to observe the protective effect of various protectivemeasures on cadaver lung; to study the protective effect and mechanismof ischemic postconditioning on ischemic reperfusion injury in rat NHBDlung transplantation.
Methods:
PartⅠ: sixty four Sprague-Dawley rats were randomly divided intofour groups, heart-beating donor group (HBD group), non-heart-beatingdonor group without protective measure on cadaver lung (NHBD-Ngroup), non-heart-beating donor group with continuous mechanicalventilation on cadaver lung (NHBD-V group), and non-heart-beatingdonor group with topical cooling on cadaver lung by ice saline (NHBD-Igroup); PartⅡ: forty Sprague-Dawley rats were randomly divided intofour groups, namely control group (NHBD-C group) and ischemicpostconditioning group (NHBD-S group) with 5 cycles of 1 min'reperfusion and 1 min' reocclusion. Surviving rate in the observationperiod of 2 hour after transplantation, lung compliance, blood gas check, the wet/dry ratio of lung after transplantation, and pathological changeswere analyzed and compared among groups in PartⅠand PartⅡ.Besides, in PartⅠ, the amount of ATP、ADP and AMP in lung tissueafter transplantation were checked by high-performance liquidchromatography(HPLC), and the sum of adenylic nucleotides werecomputerized and compared among each group; in PartⅡ, the amount ofSOD and MDA, the expression of cell apoptosis and ICAM-1mRNA inlung tissue after transplantation were assessed, and ultramicroscopicstructural changes also observed in PartⅡ.
Results: (in PartⅠand PartⅡ)
1. Surviving rate: in PartⅠand PartⅡ, each group had highsurviving rate and there were no difference between each group.
2. Lung compliance: in PartⅠ, the lung compliance in NHBD-Ngroup was much worse than that in HBD group, and there wereno significant difference among HBD group、NHBD-V groupand NHBD-I group; in PartⅡ, the lung compliance in NHBD-Sgroup was much better that in control group.
3. Blood gas check: in PartⅠ, there were no significant differencebetween each group; in PartⅡ, the PaO2 and OI were higher inNHBD-S group compared with NHBD-C group.
4. The wet/dry ratio of lung: in PartⅠ, the wet/dry ratio of lung inNHBD-N group was much higher than that in other groups, there were no significant difference among HBD group、NHBD-Vgroup and NHBD-I group; in PartⅡ, the wet/dry ratio of lung inNHBD-S group was much lower than that in NHBD-C group (p＜0.01).
5. The amount of ATP、ADP and AMP: in PartⅠ, the amount ofATP、ADP and AMP and TAN in HBD group＞that in NHBD-Igroup＞that in NHBD-V group＞that in NHBD-N group.
6. Pathological changes: in PartⅠ, the lung histologicalexamination showed that pathological changes in NHBD-Ngroup and in NHBD-V group were more serious than that inHBD group, and the pathological changes in NHBD-I group wasclose to that in HBD group; in PartⅡ, the pathological changesin NHBD-C group were more serious than that in NHBD-Sgroup.
7. The expression of cell apoptosis: in PartⅡ, the expression of cellapoptosis in NHBD-C group were much higher than that inNHBD-S group.
8. The amount of SOD and MDA: in PartⅡ, the amount of SOD inlung tissue after transplantation in NHBD-S group was muchhigher than that in NHBD-C group; the amount of MDA in lungtissue after transplantation in NHBD-S group was much lowerthan that in NHBD-C group.
9. The expression of ICAM-1mRNA: assessed by RT-PCR, theexpression of ICAM-1mRNA in lung tissue after transplantationin NHBD-S group was obviously down-regulated compared withNHBD-C group.
10.Ultramicroscopic structural changes: Under electronicmicroscope, the ultramicroscopic structural changes in NHBD-Cgroup was more serious than that in NHBD-S group.
Conclusion:
1. Establishing animal model of non-heart-beating donor in rat lungtransplantation with two-cuff-one-stent technique is a simple andeffective method, which is fit for studying early problems inNHBD lung transplantation.
2. The NHBD lung with 30 min of WIT is suitable for lungtransplantation in rats; and routine heparinization in donor andreceptor is not necessary.
3. Excellent protective effect on cadaver lung can be reached bycontinuous ventilation or topical cooling, and the latter is a litterbetter than the former.
4. Ischemic postconditioning has protective effect on NHBD lungtransplantation, it may decrease ischemic reperfusion injury afterlung transplantation and preserve the structure and function oflung.
5. The mechanism of ischemic postconditioning includes: increasethe amount of SOD and decrease the amount of MDA; inhibit theexpression of cell apoptosis after transplantation; down-regulatethe expression of ICAM-1mRNA of lung tissue aftertransplantation.
引文
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