缺血预处理在肝脏外科中的应用

近年来缺血预处理已经从动物实验阶段走向临床应用，但其如何保护肝脏拮抗热缺血再灌注损伤或移植肝在保存过程中冷缺血损伤的机制还未完全明确，在肝脏外科临床的应用中也还存在不同观点。文中就缺血预处理在肝脏外科临床中的应用情况作一综述。     

Advantage of ischemic preconditioning for hepatic resection in pigs

BACKGROUND: Ischemic preconditioning (IP) and intermittent inflow occlusion (IO) have provided beneficial outcomes in hepatic resection. However, comparison of these two procedures against warm hepatic ischemia-reperfusion injury has not been studied enough. MATERIALS AND METHODS: Pigs that had undergone 65% hepatectomy were subjected to Control (120 min continuous ischemia, n = 6), IP (10 min ischemia and 10 min reperfusion, followed by 120 min continuous ischemia, n = 6), and IO (120 min ischemia in the form of eight successive periods of 15 min ischemia and 5 min reperfusion, n = 6). We evaluated hepatocyte injury by aspartate aminotransferase, lactate dehydrogenase and hepaplastin test, hepatic microcirculation by hepatic tissue blood flow (HTBF) and endothelin (ET)-1, inflammatory response by tumor necrosis factor-alpha (TNF-alpha), and histopathology after reperfusion. RESULTS: IP prevented hepatocyte injury, HTBF disturbance, and hepatocyte necrosis in histopathology as well as IO. These two groups showed significantly better outcomes than Control. IP produced significantly less ET-1 and TNF-alpha than IO. CONCLUSIONS: IP ameliorated hepatic warm ischemia-reperfusion injury. Furthermore, IP gained more advantages in preventing chemokine production such as ET-1 and inflammatory response over IO. IP could take the place of IO for hepatectomy.     

缺血预处理对缺血再灌注损伤大鼠肾组织TLR2表达的影响

目的观察缺血预处理对缺血／再灌注损伤大鼠肾组织Toll样受体2（TLR2）表达的影响。方法将24只雄性SD大鼠随机分为3组：假手术对照组（SHAM组）、单纯缺血再灌注组（I／R组）和缺血预处理组（IPC组）,每组8只。于术后24h留取各组大鼠血标本和肾组织。检测各组大鼠血肌酐（SCr）和尿素氮（BUN）,肾组织切片行HE染色后观察其病理学改变,酶联免疫吸附法（ELISA）检测肾组织肿瘤坏死因子-α（TNF-α）含量,实时荧光定量多聚酶链反应检测肾组织TLR2mRNA表达,Westernblot检测肾组织TLR2蛋白表达。结果与SHAM组相比,I／R组大鼠SCr和BUN升高,肾组织TNF-α含量和TLR2蛋白表达升高,差异均有统计学意义（P〈0．01）;与I／R组相比,IPC组大鼠SCr和BUN降低,肾组织TNF-α含量和TLR2蛋白表达降低,差异均有统计学意义（P〈0．05）。结论缺血预处理可能通过抑制大鼠。肾组织TLR2表达及其信号通路,下调TNF-α的表达,发挥其肾脏保护作用。     

肝切除及肝移植术中缺血预处理的临床研究进展

缺血再灌注损伤一直是肝切除及肝移植术中存在的问题,是术后肝功能衰竭和移植物无功能的主要原因,而缺血预处理能减轻这种损伤。文中综述了近年来缺血预处理及其在临床应用中的研究进展,探讨了该技术在临床上的实用价值。     

不同预处理对大鼠肝移植供肝保护作用的探讨

目的 通过应用缺血、加热等多种手段对大鼠供肝进行移植术前的预处理,比较各种预处理方法对大鼠肝移植供肝缺血再灌注损伤的保护作用. 方法 将SD大鼠50只,随机分为5组:半肝缺血预处理组、脾脏缺血预处理组、热休克预处理组、热休克+缺血预处理组及手术对照组,分别进行肝脏预处理后行模拟原位肝移植术,术后检测胆汁流量,术后24 h检测血清ALT,AST,ALP水平并观察肝脏形态学变化. 结果 半肝缺血预处理组、热休克预处理组移植后胆汁分泌量多于对照组,血清ALT,AST水平明显低于对照组(P<0.05);热休克+缺血预处理组的血清ALT水平低于对照组(P<0.05),胆汁分泌量及血清AST与对照组没有显著差异;脾脏缺血预处理组的胆汁分泌量多于对照组,血清ALT水平低于对照组(P<0.05). 结论 肝脏缺血预处理初始阶段保护作用最明显,将缺血及热休克预处理两种方法联合处理大鼠时,其保护作用弱于单独缺血或单独热休克的预处理方法;脾脏缺血预处理也具有保护肝脏的作用.     

肝脏缺血预处理在肝癌围手术期中的作用

目的 观察缺血预处理(ischemic preconditioning,IP)在肝癌围手术期的作用.方法 IP组35例,对照组25例,IP组肝切除前采用10min/10 min的缺血预处理,肝门阻断方法(Pringle手法)、肝切除方法与步骤与对照组类似,比较两组术前、术后肝酶的变化、术中出血与输血量、术后并发症、住院天数、住院费用的区别.结果 两组术中肝门阻断时间、出血量、输血量无明显区别(P>0.05).术后两组肝酶均有明显升高,但IP组谷丙转氨酶在术后1 d、3 d、7 d的升高值明显低于对照组(P<0.025),IP组术后肝功能不全的发生率、住院天数和住院费用明显较对照组低(P<0.05).结论 肝癌手术时采用缺血预处理为一简单有效的保护肝功能的方法.     

Role of ischemic preconditioning in hepatic ischemia-reperfusion injury

Background

Investigation into less traumatic method of vascular occlusion during liver resection is the actual problem in hepatic surgery because of high level of complications such as liver failure. In this connection, the goal of our study was to determine the optimal model of vascular clamping. The research showed that vascular occlusion with ischemic preconditioning in the mode 5/10/15 the most delicate technique.

Methods

Forty white giant rabbits were divided randomly into four groups (n=10 in each group). In group I we used continuous Pringle maneuver by 30 min. In group II we used intermittent Pringle maneuver: 15 min of clamping/5 min of unclamping (reperfusion)/15 min of clamping. In group III we used intermittent Pringle maneuver with ischemic precondition: 5 min of ischemia/5 min of reperfusion, 10 min of ischemia/5 min of reperfusion/15 min of ischemia. Group IV (control group) is without hepatic ischemia. All animals were performed a liver biopsy at the end of the surgery. Five rabbits from each group underwent re-laparotomy on day 3 after surgery with biopsy samples being taken for studying reparative processes in liver parenchyma.

Results

Results of morphometric analysis were the best to illustrate different level of liver injury in the groups. Thus, there were 95.5% damaged hepatocytes after vascular occlusion in hepatic preparations in group I, 70.3% damaged hepatocytes in group II, and 42.3% damaged hepatocytes in group III. There were 5.3% damaged hepatocytes in the control group.

Conclusions

Vascular occlusion with ischemic preconditioning in the mode 5/10/15 the most delicate technique that does not involve major structural injuries and functional disorders in the remnant liver. Thus, it is amenable to translation into clinical practice and may improve outcomes in liver resection with inflow vascular occlusion.     

Multivariate analysis of liver regenerative capacity after hepatectomy in humans

Many factors affect liver regeneration after partial hepatectomy; however, those factors that are essential for regulation of liver regeneration in humans are not known. Using multiple regression analysis we conducted a study to determine essential factors involved in the speed of liver regeneration after hepatectomy. The subjects were 59 patients who underwent hepatic resection between January 1980 and December 1991. A regression equation for predicting regeneration speed (Y; cm³/day) during the 1st postoperative month was obtained by stepwise forward multiple regression analysis, using 11 explanatory parameters (Xi). The regeneration speed and the resection ratio (%; indicating the magnitude of resection) were calculated based on a computed tomography (CT) scan volumetric study. The degree of liver fibrosis, expressed as the fibrotic index (%), was morphometrically determined in Azan-Mallory stained sections. Of the 11 explanatory parameters, the resection ratio and the fibrotic index had a significant simple correlation with Y. The following regression equation was obtained: Y (cm³/day)=–1.1+3.7 × resection ratio –5.4 × alkaline phosphatase –3.7 × fibrotic index +1.2× total bilirubin –2.6 × glutamic pyruvic transaminase (multiple correlation coefficient, 0.82). We found that the extent of resection and the degree of fibrosis, as well as alkaline phosphatase, total bilirubin, and glutamic pyruvic transaminase, contributed to the speed of regeneration after partial hepatectomy.     

缺血预处理对大鼠肝移植受体的保护性作用

目的:观察缺血预处理对肝移植后早期C-X-C类趋化因子的表达和中性粒细胞浸润的影响,以探讨其保护作用的机制.方法:建立大鼠肝移植模型,供肝于UW液保存24 h.实验分大鼠肝移植组(未处理组)和缺血预处理组,后者于供肝切取前夹闭第一肝门10 min然后开放10 min取肝.于移植后1 h、2 h、4 h、6 h分别测定肝内髓过氧化物酶(myeloperoxidase,MPO)活性和血清肝酶学指标、巨噬细胞炎性蛋白(macrophage inflammatory protein,MIP)-2、肿瘤坏死因子(tumor necrosis factor,TNF)-α水平.分别取术后4 h肝组织行常规病理切片.结果:肝酶学指标改变显示缺血预处理组肝功能得到有效改善.缺血预处理组较未处理组肝MPO水平在各时点均有降低,并在移植后4 h(0.48±0.18 U/g组织和0.85±0.11 U/g组织)和6 h(0.63±0.04 U/g组织和0.77±0.05 U/g组织)出现显著性差异(P<0.05).血清MIP-2水平亦在4 h和6 h显著下降(2546.59±707.78 pg/ml和3488.82±785.15 pg/ml,1023.72±656.86 pg/ml和1852.04±1108.89 pg/ml).血清TNF-α水平仅在2 h变化显著(370.77±146.82 pg/ml和517.41±57.30 pg/ml).结论:本研究认为缺血预处理可能通过下调肝细胞对趋化因子的表达,减少移植肝内中性粒细胞的浸润,从而对供肝起保护作用.     

不同时限缺血预处理对硬化肝脏保护作用的实验研究

目的: 探讨缺血预处理对硬化肝脏缺血再灌注的作用,并寻找一种有效缺血预处理的时间窗和理想方案. 方法: 将64只雄性、肝硬化SD大鼠随机分为八组,每组八只:假手术组(SO组);缺血再灌注组(I/R组);缺血预处理1、2、3、4、5和6组(IPC1、IPC2、IPC3、IPC4、IPC5和IPC6).以肝组织ATP、ADP、AMP及EC(用高效液相色谱法测定),血清ALT、AST、LDH(用全自动生化仪测定)和肝脏胆汁分泌量来评价肝功能. 结果: 再灌注末,IPC3组、IPC4组、IPC5组ATP含量均明显高于I/R组(P分别为0.01、0.07和0.000);同样,测定EC时发现,IPC3组、IPC4组和IPC5组均明显高于I/R组(P=0.000).与I/R组比较,IPC4组和IPC5组的血清ALT差异有显著性(P分别为0.013和0.000);其血清LDH差异亦有显著性(P=0.023,P=0.000),而血清AST却只有IPC5组显著低于I/R组(P=0.001).IPC3组、IPC4组和IPC5组肝脏的胆汁分泌量明显高于I/R组(P=0.028,P=0.023,P=0.008). 结论: 5~10min予一次或两次缺血预处理,能启动IPC对肝硬化大鼠肝脏I/R损伤的保护作用;10 min的缺血预处理,对肝硬化大鼠肝脏I/R损伤的保护作用最强.     

Protective effect of ischemic preconditioning against intermittent warm-ischemia-induced liver injury

BACKGROUND: Although ischemic preconditioning (IPC) has been reported to protect the liver from injury when subjected to continuous hepatic ischemia, whether IPC protects rat livers against ischemia-reperfusion (I/R) injury after intermittent ischemia has not been elucidated. MATERIALS AND METHODS: Five groups of Wistar rats were subjected to intermittent hepatic ischemia (I) comprising 15-min ischemia and 5-min reperfusion three times with or without prior IPC (10-min ischemia and 10-min reperfusion), 45-min continuous ischemia (C) with or without IPC, and sham operation. Serum transaminase and lactic acid levels, hepatic tissue energy charges, and hepatic blood perfusion were measured after reperfusion. Plasma tumor necrosis factor-alpha (TNF-alpha) levels were determined after reperfusion for 120 min. Histological and apoptotic findings were evaluated after reperfusion for 180 min. RESULTS: IPC significantly reduced serum transaminase levels after continuous and intermittent ischemia (IPC + C, 1107 vs C, 2684 IU/l; IPC + I, 708 vs I, 1859 IU/l). After hepatic ischemia without IPC, apoptosis and necrosis with increased plasma TNF-alpha levels were observed. IPC protected livers from injury by interfering with the increase in plasma TNF-alpha (IPC + I, 27.6 vs I, 64.8 pg/ml; IPC + C, 21.6 vs C, 49.3 pg/ml). This resulted in the attenuation of hepatic necrosis after continuous ischemia and significantly reduced necrosis and apoptosis after intermittent ischemia. CONCLUSIONS: IPC exerts a greater protective effect against hepatic I/R injury after intermittent hepatic ischemia than after continuous hepatic ischemia.     

不同时间的缺血预处理对肝硬化大鼠缺血再灌注损伤的保护作用

目的 探讨缺血预处理(IPC)对肝硬化大鼠缺血再灌注损伤(I/R)的保护作用,并寻找对肝硬化I/R保护作用的最佳有效时间窗和理想方案.方法 通过构建正常大鼠及肝硬化大鼠模型,以正常肝脏I/R(A组)和正常肝脏10-10 min-IPC方案(B组)为对照组,肝硬化组则分别施行单纯I/R(C组)、5-10 min(D组)、8-10min(E组)、10-10 min(F组)及15-10 min(G组)的IPC方案,每组18只,分别在术后1 h、4 h及24 h三个时间点采静脉血,检测血清ALT、AST、LDH及肝组织中MDA、MPO、NO、SOD水平,评价肝功能及肝脏组织炎性浸润及抗损伤程度.结果 肝脏缺血30 min后肝脏功能受损明显,表现为各组的ALT、AST、LDH水平升高,尤以再灌注4 h时显著(P<0.05),但经过缺血预处理后,各IPC组中的NO、MDA、MPO及SOD水平亦显著高于其对应的I/R组,以E组的差别有显著意义(P<0.05).结论 10-10 min的IPC方案对于正常肝脏I/R确有保护作用,而8-10 min的IPC方案能最有效地启动对肝硬化大鼠肝脏I/R的保护作用.     

缺血预处理对大鼠肝脏缺血/再灌注早期核因子κB活性及细胞凋亡的影响

目的 观察缺血预处理对大鼠肝脏缺血/再灌注早期核因子KB活性、促凋亡基因Fas和FasL蛋白表达以及肝细胞凋亡的影响,以进一步阐明肝脏缺血预处理的抗凋亡作用机制.方法 建立SD大鼠肝缺血(40min)再灌注(120 min)损伤及肝缺血预处理的模型.24只大鼠随机分成3组(n=8).①假手术对照组(C组),仅分离肝十二指肠韧带,不阻断肝门,不进行其他干预处理.②缺血再灌注组(IR组),在第一肝门用小血管夹阻断尾状叶及左肝叶血流40 min松开血管夹肝脏再灌注2 h,再灌注开始后关腹.③缺血预处理组(IP组),先行3个循环的缺血预处理,阻断第一肝门10 min,开放再灌注10 min为1个循环,随后操作同缺血/再灌注组.实验结束后全自动生化分析仪检测血清谷草转氨酶和血清谷丙转氨酶活性:TUNEL法检测肝组织的细胞凋亡指数(AI):EMSA法测定肝组织核因子κB的结合活性:Western blotting免疫印迹法检测Fas及FasL蛋白的表达水平.结果 IR组和IP组的ALT、AST及细胞凋亡指数(AI)均明显高于S组(P<0.01),但与IR相比,IP组则明显较低(P<0.01).与C组比较,IR组的核因子κB活性、促凋亡基因Fas和FasL蛋白表达水平明显增高,而IP组仅轻度增高.结论 缺血预处理可通过降低肝脏缺血/再灌注早期核因子κB的转录活性,并下调促凋亡基因Fas及FasL的表达,从而发挥抗细胞凋亡和损伤保护效应.     

Serum endostatin levels and regenerative capacities of normal and cirrhotic livers following partial hepatectomy in mice: the response to different resection sizes

BACKGROUND: Angiogenesis has an important role in liver regeneration. Antiangiogenic response in remnant liver following resection and its relationship to regeneration is not well known. The aim of this study was to investigate the effect of hepatectomy size on serum endostatin levels, and the effect of endostatin levels to liver regeneration after partial hepatectomy in normal and cirrhotic mice. MATERIALS AND METHODS: Sixty noncirrhotic and 36 carbon tetrachloride-induced cirrhotic mice were included in the study. Noncirrhotic mice were randomly divided into four main groups: sham, 20%, 40%, and 70% hepatectomy groups. Similarly, cirrhotic mice were randomly divided into three main groups: sham, 20%, and 40% hepatectomy groups. The mice in each group were further divided into two subgroups to compare serum endostatin levels and liver regeneration indexes on days 1 and 14. Liver regeneration was evaluated by the proliferating cell nuclear antigen-labeling index. Serum endostatin level was measured to evaluate antiangiogenic response. RESULTS: Serum endostatin levels on the first day and 14th day increased significantly in correlation with the hepatectomy size, both in normal mice and cirrhotic mice (P < 0.05). In normal mice with high regeneration indexes that underwent 40% and 70% hepatectomies, there was a significant increase in serum endostatin levels on the 14th day compared with the first day (P < 0.05). However, the increase in mice that underwent 20% hepatectomies was not significant. After 20% and 40% hepatectomies, first day serum endostatin levels were significantly higher in cirrhotic mice compared with normal mice (P < 0.05), which was independent of regeneration. Nevertheless, after 40% hepatectomies, 14th day serum endostatin levels were significantly lower in cirrhotic mice compared with normal mice, attributable to the limited regeneration capacity of cirrhotic liver (P < 0.05). Regeneration capacity of cirrhotic liver was low at all times. CONCLUSIONS: The current study suggests that there is a significant relationship between serum endostatin levels and regeneration capacity after hepatectomy in normal mice. On the other hand, following resection of cirrhotic liver, regeneration capacity is depressed and high endostatin levels are independent of hepatic regeneration.     

Prevention of I/R injury in fatty livers by ischemic preconditioning is associated with increased mitochondrial tolerance: the key role of ATPsynthase and mitochondrial permeability transition

Ischemia/reperfusion (I/R) injury is a commonly encountered clinical problem and occurs probably as a consequence of irreversible mitochondrial injury. The increased susceptibility of fatty livers to ischemic injury is associated with depletion of adenosine triphosphate (ATP) content, which is preserved by preconditioning. Mitochondria being the main ATP production source for the cell, we aimed to evaluate whether ischemic preconditioning (IPC) of fatty livers prevents the impairment in mitochondrial function induced by I/R. Lean and steatotic animals were subjected to 90 min of hepatic warm ischemia and 12 h of reperfusion. IPC effect was tested in fatty livers. After reperfusion, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Mitochondrial membrane potential, mitochondrial respiration and susceptibility to mitochondrial permeability transition (MPT) were evaluated, as well as ATPase activity and adenine nucleotides. IPC of fatty livers decreased serum AST and ALT levels. Fatty animals subjected to I/R exhibited decreased mitochondrial membrane potential and a delay in the repolarization after a phosphorylation cycle, associated with increased state 4 respiration. Increased tolerance to MPT induction, preservation of F₁F_o-ATPsynthase activity and mitochondrial bioenergetics were observed in ischemic preconditioned fatty livers. Thus, IPC is an endogenous protecting mechanism that preserves mitochondrial function and bioenergetics in fatty livers.     

The effect of ischemic preconditioning prior to intraoperative radiotherapy on ischemic and on reperfused rat liver

BACKGROUND: The purpose of this study was to increase the tolerance of the liver to radiation injury with the proven effect of ischemic precondition (IP) in decreasing oxygen-derived free radicals, and to compare the effect of intraoperative radiotherapy (IORT) during ischemia and during reperfusion on rat liver. MATERIALS AND METHODS: Two hundred fifty to 280 g male Wistar rats underwent 45 min of normothermic, segmental liver ischemia with or without IP/5 min ischemia and 10 min reperfusion, in two cycles. During ischemia or reperfusion, IORT doses of 0, 25, or 50 Gy were applied to the ischemic liver lobe. Hepatic microcirculation was monitored by laser Doppler flowmeter. Short- and long-term histological, alkaline phosphatase, bilirubin and tumor necrosis factor-alpha levels, liver tissue, and serum antioxidant alterations were measured. RESULTS: Histological, laboratory, as well as flowmetry alterations caused by 25 Gy were reversible after 6 mo. Three mo following IORT, histological examination revealed parenchymal fibrosis, bridging, liver cell atrophy, and bile duct proliferation in the group that was irradiated with 50 Gy during reperfusion, without IP. In this group, the changes were present 6 mo following IORT, and also the levels of tumor necrosis factor-alpha and oxygen-derived free radicals after reperfusion were increased. All these changes were significantly milder in groups with IP, especially those that were irradiated during ischemia. CONCLUSIONS: IORT to the liver, up to 25 Gy, can be applied without short- or long-term treatment morbidity. Doses of up to 50 Gy are tolerated with IP, which has never been described before. Irradiation during ischemia is less toxic for the liver tissue.     

慢性前列腺炎患者前列腺液IL-1β和TNF-α的检测及意义

目的 :了解IL 1β和TNF α在慢性前列腺炎患者前列腺按摩液 (EPS)中的变化及临床意义。 　方法 :应用ELISA法对 34例慢性前列腺炎 [EPS中白细胞 (WBC)计数≥ 10 /HP为A组 16例 ,WBC <10 /HP为B组 18例 ]、10例无症状性前列腺炎、12例良性前列腺增生 (BPH)及 8例健康对照EPS中的IL 1β和TNF α进行检测。 　结果 :IL 1β和TNF α在EPS中WBC≥ 10 /HP的慢性前列腺炎和无症状性前列腺炎两组检测值明显高于WBC <10 /HP的慢性前列腺炎、BPH和健康对照 3组 (P <0 .0 5 ,P <0 .0 2 )。IL 1β与TNF α有显著的数列等级相关性 (P <0 .0 0 3) ,而WBC计数和IL 1β、TNF α之间的数列等级相关性无显著性意义。 　结论 :IL 1β与TNF α在伴有WBC计数增高的慢性前列腺炎患者EPS中明显增高 ,IL 1β与TNF α对传统以WBC计数为慢性前列腺炎进行分类的方法可以提供一个更准确的新分类方法。     

缺血预处理对肢体缺血再灌注损伤的影响

目的　观察缺血预处理 (IPC)对肢体缺血再灌注损伤的影响。方法　选择 2 0例需充气止血带止血进行手术的患者 ,随机分为对照组 (n =10 )和IPC组 (n =10 )。IPC组患者术前应用 3次 5min循环缺血 ,间隔 5min再灌注预处理后在止血带下进行手术 ;对照组直接在止血带下进行手术。在肢体缺血前和再灌注 30min、90min、180min分别取静脉血检测血清肌酸磷酸激酶 (CPK)、谷草转氨酶(AST)、乳酸脱氢酶 (LDH)、丙二醛 (MDA)和过氧化物歧化酶 (SOD)水平。结果　随着肢体缺血再灌注时间的延长 ,血中CPK、AST、LDH、MDA含量逐渐升高 ,而SOD活性逐渐降低。IPC组在缺血前及再灌注同时间 ,血中CPK、AST、LDH、MDA含量低于对照组 (P <0 0 5 ,P <0 0 1) ;而SOD活性高于对照组 (P <0 0 5 ,P <0 0 1)。结论　IPC能有效地减轻肢体缺血再灌注损伤程度 ,减轻脂质过氧化反应 ,提高肢体缺血耐受性