实验性原位肝移植供肝热缺血损伤研究
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摘要
目的 探讨原位肝移植中供肝热缺血再灌注损伤的发生机制及其影响。
方法 本实验共分4组，其中包括对照组(C)和供肝热缺血0分钟(W_0)、热缺血5分钟(W_5)、热缺血10分钟(W_(10))肝移植组。对照组只行胆道外引流术，肝移植各组供肝获取后均于4℃UW液保存6小时后行动脉化双套管法原位肝移植术，每组均为6只大鼠。各组均于术后24小时获取肝组织标本及取血、胆汁，所获肝标本行常规组织学检查，利用RT-PCR方法检测各组肝标本中细胞间粘附分子-1mRNA的转录水平，测定血清肝功能和胆汁酶学的变化，并测定血清内皮素-1(ET-1)浓度、超氧化物歧化酶(SOD)的活力、丙二醛(MDA)的浓度、一氧化氮(NO)的含量。
结果 常规组织病理检查显示：C组与W_0组肝小叶结构正常，肝细胞无肿胀、变性及坏死，肝窦、中央静脉未见充血，无中性粒细胞浸润。W_5组中央静脉淤血，肝窦扩张充血，肝细胞肿胀，未见坏死，无中性粒细胞浸润。W_(10)组肝细胞呈气球样变及少量坏死，以中央静脉周围明显，并可见中性粒细胞浸润。与对照组相比，肝移植组血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)、γ-谷氨酰转肽酶(GGT)、直接胆红素(DBIL)显著升高，并且随着供肝热缺血时间的延长升高，与供肝热缺血时间呈明显正相关(不包括ALP)，而肝移植组血清胆碱脂酶(CHE)、总胆红素(TBIL)变化不明显。与对照组相比，肝移植组胆汁中的GGT和TBIL无显著性差异，但胆汁中的ALT、AST、LDH在移植组中均升高，但ALT、LDH、AST的升高与供肝热缺血时间无关。与对照组相比，血清丙二醛浓度、一氧化氮含量、内皮素-1浓度移植各组中均明显增高，并且丙二醛浓度、一氧化氮含量随供肝热缺血时间的延长增加，并与热缺血时间成正相
天津医科大学硕士研究生论文摘要
关，而内皮素一1在移植各组间无明显差异。与对照组相比血浆超氧化物歧化
酶活力在移植各组中均下降，但与供肝热缺血时间无关。移植组的
ICAM一lmRNA的转录水平均较对照组明显增高;并且随着供肝热缺血时间
的延长，ICAM一1 mRNA的转录水平明显增加，并与热缺血时间成正相关。
结论近交系大鼠动脉化原位肝移植模型能过很好的反映肝移植过程中
的供肝热缺血损伤，更适合肝移植缺血再灌注损伤的研究。肝移植过程中供
肝热缺血主要损伤肝细胞，并随着供肝热缺血时间的延长，移植肝细胞损伤
加重，并且血清中的ALT、AST、LDH可能反映出供肝热缺血造成肝细胞损
伤的程度，可作为肝移植过程中供肝热缺血损伤的指标。血清GGT及DBIL
可反映胆汁淤积的程度。氧自由基、ET一1、NO、ICAM一1参与供肝热缺血
造成的移植肝损伤，供肝的热缺血可通过增加氧自由基形成和再灌注晚期NO
生成增加加重缺血再灌注损伤过程，加重移植肝损伤。ET一1可能反映供肝冷
保存的损伤。供肝热缺血可通过再灌注晚期增加ICAM一lmRNA表达，使
ICAM一1生成增加加重移植肝的损伤。ICAM一l可能是导致移植肝缺血再灌注
时肝细胞损伤的主要因素，是反映肝细胞损伤的预警指标。
Objective To study the mechanism and effection due to warm ischemia reperfusion injury in graft following orthotopic liver transplantation.
Methods The rats were divided into four groups (n=6/group) including the control group that biliary extra-drainage was only performed and 3 liver transplantation groups that warm ischemia time was respectively 0, 5, 10 min (W0,W5,Wio). In all transplantation groups, liver grafts were stored in UW solution (4C) for 6 hours before implantation. Orthotopic liver transplantation with hepatic artery reconstruction and biliary extra-drainage model using inbred male BN rats was established under microscopy. At 24 hours postoperatively, liver samples were collected for histopathological studies and determining the mRNA transcription levels of ICAM-1 with quantitative reserve transcription chain reaction (QRT-PCR). Blood samples were taken simultaneously for measurement of hepatic function, nitric oxide (NO), endothelin-1 (ET-1), malondialdehyde (MDA), superoxide dismutase (SOD). Meanwhile bile was also gotten to assay lactate dehydrogenase(LDH), alanine transaminase(ALT), aspartate transaminase(AST), Y -glutamyltran
sferase(GGT) and total bilirubin(TBIL).
Results Histopathological studies showed remarkable morphological changes were not observed in rats of Group C and Group W0. It was also showed normal hepatic parenchyma and not discovered that damage of hepatocyte, sinusoidal congestion, and neutrophils infiltrating into hepatic tissue. In Group W5, congested
in central vein of hepatic lobule and sinusoid and swelling hepatocyte were exhibited. There were no hepatocytes necrosis and infiltration of neutrophils. It was discovered ballooning hepatocytes, hepatocytes necrosis and neutrophils infiltrating in Group WIQ- Compared with C group, serum AST, ALT, LDH, GGT, alkaline phosphatase(ALP), direct bilirubin(DBIL) were markedly increased in liver transplantation group and positively correlated with liver graft warm ischemia time except for ALP. The serum cholinesterase(CHE) and TBIL were not siginifiantly altered in each group. Compared with C group, ALT,AST, LDH in bile were markedly increased in liver transplantation groups without correlation to warm ischemia time. There was no significantly changing of GGT and TBIL in bile of four groups. In liver transplantation groups, Serum MDA, NO and ET-1 were significantly increased, but SOD activity was decreased markedly. Among those markers, MDA and NO were related with the warm ischemia time, while ET-1 and SOD were not related. Compared with Group C, the mRNA transcription level of ICAM-1 was also marked increasing in liver transplantation groups and positive correlation with graft warm ischemia time.
Conclusion Orthotopic liver transplantation with artery reconstruction in inbred rats can embody the warm ischemia of graft in liver transplantation and might be suitable for research on ischemia-reperfusion injury post liver transplantation. Hepatocyte is the main site of warm ischemia injury. With increasing warm ischemia time of graft, the injury of liver graft became crisis. It is demonstrated that serum AST, ALT, LDH could present the degree of warm ischemia injury and might be valuable markers serving as predicting index. Associated with the increasing warm ischemia of graft, cholestasis aggravated with elevating serum GGT and TBIL lever. It plays the key role in warm ischemia injury of graft after liver transplantation that are MDA, ICAM-1, ET-1 and NO. The warm ischemia of graft heightened production of oxygen-derived free radicals. The warm ischemia injury of liver was aggravated with NO and ICAM-1 in the later stage of reperfusion. ET-1 could reflect the cold ischemia injure of graft in liver transplantation. ICAM-1 may be a good marker to indicate the degree of
hepatocyte injure.
方法 本实验共分4组，其中包括对照组(C)和供肝热缺血0分钟(W_0)、热缺血5分钟(W_5)、热缺血10分钟(W_(10))肝移植组。对照组只行胆道外引流术，肝移植各组供肝获取后均于4℃UW液保存6小时后行动脉化双套管法原位肝移植术，每组均为6只大鼠。各组均于术后24小时获取肝组织标本及取血、胆汁，所获肝标本行常规组织学检查，利用RT-PCR方法检测各组肝标本中细胞间粘附分子-1mRNA的转录水平，测定血清肝功能和胆汁酶学的变化，并测定血清内皮素-1(ET-1)浓度、超氧化物歧化酶(SOD)的活力、丙二醛(MDA)的浓度、一氧化氮(NO)的含量。
结果 常规组织病理检查显示：C组与W_0组肝小叶结构正常，肝细胞无肿胀、变性及坏死，肝窦、中央静脉未见充血，无中性粒细胞浸润。W_5组中央静脉淤血，肝窦扩张充血，肝细胞肿胀，未见坏死，无中性粒细胞浸润。W_(10)组肝细胞呈气球样变及少量坏死，以中央静脉周围明显，并可见中性粒细胞浸润。与对照组相比，肝移植组血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)、γ-谷氨酰转肽酶(GGT)、直接胆红素(DBIL)显著升高，并且随着供肝热缺血时间的延长升高，与供肝热缺血时间呈明显正相关(不包括ALP)，而肝移植组血清胆碱脂酶(CHE)、总胆红素(TBIL)变化不明显。与对照组相比，肝移植组胆汁中的GGT和TBIL无显著性差异，但胆汁中的ALT、AST、LDH在移植组中均升高，但ALT、LDH、AST的升高与供肝热缺血时间无关。与对照组相比，血清丙二醛浓度、一氧化氮含量、内皮素-1浓度移植各组中均明显增高，并且丙二醛浓度、一氧化氮含量随供肝热缺血时间的延长增加，并与热缺血时间成正相
天津医科大学硕士研究生论文摘要
关，而内皮素一1在移植各组间无明显差异。与对照组相比血浆超氧化物歧化
酶活力在移植各组中均下降，但与供肝热缺血时间无关。移植组的
ICAM一lmRNA的转录水平均较对照组明显增高;并且随着供肝热缺血时间
的延长，ICAM一1 mRNA的转录水平明显增加，并与热缺血时间成正相关。
结论近交系大鼠动脉化原位肝移植模型能过很好的反映肝移植过程中
的供肝热缺血损伤，更适合肝移植缺血再灌注损伤的研究。肝移植过程中供
肝热缺血主要损伤肝细胞，并随着供肝热缺血时间的延长，移植肝细胞损伤
加重，并且血清中的ALT、AST、LDH可能反映出供肝热缺血造成肝细胞损
伤的程度，可作为肝移植过程中供肝热缺血损伤的指标。血清GGT及DBIL
可反映胆汁淤积的程度。氧自由基、ET一1、NO、ICAM一1参与供肝热缺血
造成的移植肝损伤，供肝的热缺血可通过增加氧自由基形成和再灌注晚期NO
生成增加加重缺血再灌注损伤过程，加重移植肝损伤。ET一1可能反映供肝冷
保存的损伤。供肝热缺血可通过再灌注晚期增加ICAM一lmRNA表达，使
ICAM一1生成增加加重移植肝的损伤。ICAM一l可能是导致移植肝缺血再灌注
时肝细胞损伤的主要因素，是反映肝细胞损伤的预警指标。
Objective To study the mechanism and effection due to warm ischemia reperfusion injury in graft following orthotopic liver transplantation.
Methods The rats were divided into four groups (n=6/group) including the control group that biliary extra-drainage was only performed and 3 liver transplantation groups that warm ischemia time was respectively 0, 5, 10 min (W0,W5,Wio). In all transplantation groups, liver grafts were stored in UW solution (4C) for 6 hours before implantation. Orthotopic liver transplantation with hepatic artery reconstruction and biliary extra-drainage model using inbred male BN rats was established under microscopy. At 24 hours postoperatively, liver samples were collected for histopathological studies and determining the mRNA transcription levels of ICAM-1 with quantitative reserve transcription chain reaction (QRT-PCR). Blood samples were taken simultaneously for measurement of hepatic function, nitric oxide (NO), endothelin-1 (ET-1), malondialdehyde (MDA), superoxide dismutase (SOD). Meanwhile bile was also gotten to assay lactate dehydrogenase(LDH), alanine transaminase(ALT), aspartate transaminase(AST), Y -glutamyltran
sferase(GGT) and total bilirubin(TBIL).
Results Histopathological studies showed remarkable morphological changes were not observed in rats of Group C and Group W0. It was also showed normal hepatic parenchyma and not discovered that damage of hepatocyte, sinusoidal congestion, and neutrophils infiltrating into hepatic tissue. In Group W5, congested
in central vein of hepatic lobule and sinusoid and swelling hepatocyte were exhibited. There were no hepatocytes necrosis and infiltration of neutrophils. It was discovered ballooning hepatocytes, hepatocytes necrosis and neutrophils infiltrating in Group WIQ- Compared with C group, serum AST, ALT, LDH, GGT, alkaline phosphatase(ALP), direct bilirubin(DBIL) were markedly increased in liver transplantation group and positively correlated with liver graft warm ischemia time except for ALP. The serum cholinesterase(CHE) and TBIL were not siginifiantly altered in each group. Compared with C group, ALT,AST, LDH in bile were markedly increased in liver transplantation groups without correlation to warm ischemia time. There was no significantly changing of GGT and TBIL in bile of four groups. In liver transplantation groups, Serum MDA, NO and ET-1 were significantly increased, but SOD activity was decreased markedly. Among those markers, MDA and NO were related with the warm ischemia time, while ET-1 and SOD were not related. Compared with Group C, the mRNA transcription level of ICAM-1 was also marked increasing in liver transplantation groups and positive correlation with graft warm ischemia time.
Conclusion Orthotopic liver transplantation with artery reconstruction in inbred rats can embody the warm ischemia of graft in liver transplantation and might be suitable for research on ischemia-reperfusion injury post liver transplantation. Hepatocyte is the main site of warm ischemia injury. With increasing warm ischemia time of graft, the injury of liver graft became crisis. It is demonstrated that serum AST, ALT, LDH could present the degree of warm ischemia injury and might be valuable markers serving as predicting index. Associated with the increasing warm ischemia of graft, cholestasis aggravated with elevating serum GGT and TBIL lever. It plays the key role in warm ischemia injury of graft after liver transplantation that are MDA, ICAM-1, ET-1 and NO. The warm ischemia of graft heightened production of oxygen-derived free radicals. The warm ischemia injury of liver was aggravated with NO and ICAM-1 in the later stage of reperfusion. ET-1 could reflect the cold ischemia injure of graft in liver transplantation. ICAM-1 may be a good marker to indicate the degree of
hepatocyte injure.
引文
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