【作者】 冯春林；

【导师】 别平；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 一、研究背景在临床上,一些恶性肿瘤如:胰头癌、胆管癌(Ⅰ型)等,常以黄疸为首发症状,肿瘤常早期侵犯门静脉,导致较低的根治性切除率。近年来,联合门静脉切除重建的胰十二指肠切除术得到了较为广泛的认同,极大地提高了这类肿瘤的切除率,但是术中门静脉血流急性阻断( portal vein occlusion,PVO )对合并有梗阻性黄疸( obstructive jaundice, OJ )的肝脏必然造成进一步损害。目前,对肝脏缺血再灌注损伤(ischemia reperfution injury,IRI)的研究较多,但对于合并有OJ的PVO对肝脏引起的IRI的研究还未见报道。因此深入研究OJ情况下,PVO后肝脏病理生理改变的分子机制以及肝脏耐受PVO的安全时限,对临床如何把握PVO时限、提高手术耐受性、安全性、促进术后恢复具有重要意义。研究认为肝脏的IRI主要以肝细胞凋亡为特征的损害,为防止肝脏的IRI,原位低温灌注技术得到了广泛的应用,大大提高了肝脏耐受缺血的时限。常用的灌注液有乳酸林格氏液、HTK液及UW液等,取得了一定减轻IRI的效果,但以往的原位低温灌注技术也有许多不足之处:1、技术操作复杂难度大,2、易引起严重低血压、肺水肿、高钾血症、酸中毒、DIC(disseminated intravascular coagulation)等并发症,3、HTK和UW液不能进入体循环。由此我们自行配制了一种自制肝保护液( self-made liver solution,SMLS )以用于合并有OJ且侵犯门静脉的肿瘤切除术中,PVO期间进行在体经门静脉持续低温灌注,观察其是否具有肝保护作用并进一步探明其可能的机制。二、目的本课题拟通过对门静脉转流下, OJ大鼠PVO期间,用4℃SMLS经门静脉在体持续灌注肝脏,观察A20mRNA及蛋白的表达,以及MAPKs的活化与线粒体凋亡途径上各相关蛋白的表达情况的关系,以期阐明OJ条件下,PVO肝脏损伤机制以及SMLS对肝保护作用及其机制。三、方法与结果1、常温下OJ大鼠PVO模型的建立及其对肝脏能量代谢的影响:正常大鼠被随机分为SO(Shame Operation)组、OJ大鼠再随机分为A组(胆道再通组)、B组(胆道再通-PVO 30min)、C组(胆道再通-PVO 60min)及D组(胆道再通-PVO 90min)。结果:D组术后一周大鼠累计病死率60%,而SO、A、B及C组病死率为0%。D组ALT、AST、TBIL在术后各时相点都显著高于SO、A、B、C组,且在术后24h仍无下降趋势(P<0.05);通过反应肝细胞线粒体能量代谢的指标观察,D组RCR、P/O、ATP值在术后各时相点都明显低于SO、A、B及C组;肝组织HE染色光镜观察即显示,D组肝组织结构严重紊乱,肝细胞广泛空泡化及坏死,炎细胞浸润,淤胆较重,明显重于其它各组。2、SMLS对OJ大鼠PVO肝损伤的保护:OJ大鼠被随机分为E组(胆道再通、门静脉周围游离、切除肝尾状叶,90 min后关腹)、F组(胆道再通、肝尾状叶分流门静脉、余肝PVO 90 min、恢复正常门静脉血流、切除肝尾状叶,关腹)、G组(F组+门静脉肝侧灌注4℃乳酸林格氏液)、H组(F组+门静脉肝侧灌注4℃SMLS );术后各组ALT、AST和TBIL水平均呈升高趋势,均在术后6 h达高峰,在术后24 h有所下降,其中F、G及H组升高较E组明显且有显著性差异(P<0.05),而F组及G组又显著高于H组,F组高于G组之间有显著性差异(P<0.05)。未缺血的E组大鼠术后因胆道再通其内毒素( ETX )水平,在术后各时相点呈逐渐下降趋势,说明胆道梗阻在本实验中是ETX增高的主要因素,而胆道再通是有利于消除ETX的重要措施;在受到PVO损伤的F组其ETX在术后各时相点呈大幅度的逐渐增高,到术后72 h亦无明显下降趋势;而在给予门静脉灌注的G组和H组在术后虽呈逐渐增高趋势,但增高的幅度不如F组大,有显著性差异(P<0.05),且G组和H组到术后24 h开始逐渐下降;而给予SMLS的H组其ETX在术后各时相点均低于给予乳酸林格氏液灌注的G组,有统计学意义(P<0.05)。肝组织HE染色光镜观察,F组病理改变明显重于其它各组;透射电镜观察,F组线粒体损伤亦明显重于其它各组;TUNEL凋亡检测,各组术后0 h即有肝细胞凋亡,但E组在术后各时相点呈逐渐下降趋势;相反,F组、G组和H组均呈逐渐升高趋势,在术后6 h达到高峰,在术后24 h有所下降,其中F组和G组明显高于H组并有显著性差异(P<0.05)。F组高于G组且有显著性差异(P<0.05)。3、MAPKs信号转导途径在SMLS保护肝细胞中的作用及机制:采用逆转录PCR、Western blotting及TUNEL等技术分别检测A20mRNA及其蛋白、P-JNK、P-ERK、P-p38、bcl-2、Bax、caspase-3等蛋白以及肝细胞凋亡百分率的检测,同时给予MAPK信号转导途径的特异性抑制剂SP600125(JNK特异性抑制剂)、PD98059(ERK特异性抑制剂)和SB203580(P-38特异性抑制剂)在灌注SMLS前30min,经尾静脉注射作为预处理,再次观察各组肝细胞凋亡的变化;结果显示,H组A20mRNA,A20、P-ERK及bcl-2蛋白显著高于E、F及G组(P<0.05),其凋亡率却相反,G组又显著高于F组(P<0.05),而F组P-JNK、Bax、caspase-3蛋白及凋亡率显著增高,与E、G及H组比有显著性差异(P<0.05),当被给予特异性抑制剂后,给予SP600125时肝细胞凋亡进一步减少,而给予PD98059时肝细胞凋亡增加,给予SB203580时肝细胞凋亡无明显变化。四、结论1、梗阻性黄疸大鼠门静脉血流急性阻断引起肝细胞线粒体能量代谢障碍;2、初步判定梗阻性黄疸大鼠在门静脉转流下,耐受门静脉血流阻断的安全时限为60 min;3、肝细胞凋亡是梗阻性黄疸条件下,门静脉血流急性阻断对肝脏造成的缺血再灌注损伤引起肝损害的一种重要方式。线粒体途径可能介导了肝细胞的凋亡;4、经门静脉在体灌注低温乳酸林格氏液或自制肝保护液,可以减轻梗阻性黄疸大鼠因门静脉血流急性阻断导致的肝细胞线粒体损伤及肝细胞凋亡。其中自制肝保护液的肝保护作用明显优于乳酸林格氏液;5、梗阻性黄疸大鼠门静脉血流急性阻断激活JNK信号转导通路,介导线粒体途径bcl-2/bax蛋白平衡的偏移,活化下游的凋亡执行蛋白caspase-3,从而促进肝细胞凋亡;6、自制肝保护液经门静脉在体持续低温灌注肝脏,可以促进A20mRNA及其蛋白的高表达,由此激活ERK信号转导通路,抑制JNK信号转导通路,反向调节线粒体途径bcl-2/bax蛋白平衡的逆转,降低了其下游的凋亡执行蛋白caspase-3的表达,从而起到抗凋亡、保护肝细胞功能的作用。更多还原

【Abstract】 BackgroundJaundice is always the most common symptom in patients with malignant tumors such as pancreatic carcinoma,cholaniocarcinoma and so on.The character of special location and early portal vein invasion always lead to low radical resection rate.In recent years,the application of pancreaticcoduodenectomy with portal vein resection in managing these cancers has been improved.But acute portal vein occlusion(PVO) can cause progressive damage to liver especially in patients with obstructive jaundice(OJ).Until now,there is no report about hepatic ischemical reperfusion injury caused by PVO in patients with OJ.Therefore ,profound research of hepatic pathophisiology changes after PVO and safe time limit to PVO in rats with OJ has important influence on promoting the safety and toleration of operation and the following recovery.Previous studies showed that apoptosis was the main character of hepatic ischemical reperfusion injury.Orthotopic hypothermical perfusion was widely used to prevent ischemical reperfusion injury that can promote hepatic tolerance of ischemia.At present,mainly used perfusion solution included Lactated Ringer’S Solution,HTK solution,UW solution and so on.These solutions can lessen ischemical reperfusion injury to some extent.But whole hepatic blood flow blockage with continuous orthotopic hypothermic perfusion also had some disadvantages:First、this technique has big difficulty and multiplicity;Second、it can cause complication such as severe hypotension, edema of lung,depression of potassium,acidosis,DIC and so on.3、HTK and UW solutions can’t exist in systemic circulation.Then we used a hepatic preservation solution(self-made liver solution,SMLS) and assessed its effect and mechanism in protecting hepatocytes .ObjectiveThe mRNA level and expression of A20 along with the relation between the activation of MAPKs and the expression of related proteins in mitochondria mediated apoptosis pathway were observed in rats with OJ and hepatic continuous hypothermic perfusion with SMLS after PVO to elucidate the mechanisms of hepatic damage by PVO in rats with OJ and protection by SMLS.Methods and results1、Animal model and effect of PVO on hepatic energy metabolism:normal rats were randomly divided into four groups:(1)group SO:shame operation;(2)group A:biliary tract recanalization;(3)group B:biliary tract recanalization with PVO 30m;(3)group C:biliary tract recanalization with PVO 60m;(3)group D: biliary tract recanalization with PVO 90m.Summary mortality of group D was 60 percent which was obviously higher than that of other groups at 1 post operation week. ALT,AST and TBIL of group D were marked higher than those of other groups and the tendency of decreasing was not showed at 24h post operation.RCR,P/O and ATP of group D were also lower than those of other groups.Hepatic tissue disorder,extensive vacuolization and necrosis of hepatocyte,infiltration of inflammatory cells and cholestasis were still obvious in group D.2、Protective effect of SMLS on hepatic function in rats with OJ and PVO:rats with OJ were randomly divided into four groups:group E(biliary tract recanalization,liberation of portal vein,resection of caudate lobe of liver and closure of abdomen at 90m ).group F(biliary tract recanalization,portal vein bypass in caudate lobe of liver,PVO of left hepatic tissue 90m,then recovery of portal vein flow and resection of caudate lobe of liver).group G(group F with perfusion of 4℃Lactated Ringer’S Solution).group H(group F with portal vein perfusion of 4C SMLS).The level of ALT,AST and TBIL of all groups were heightened post operation and got to peak at 6h post operation,and then declined after 24h post operation.The increasing value of ALT,AST and TBIL in group F,group G and group H were obviously higher than that of group E.Because of no PVO,the level of ETX of group E gradually descended which means obstruction of biliary tract is the main reason for increasing of ETX. and biliray tract recanalization is of advantage to decrease ETX.In group F,the level of ETX markedly increased post operation and no tendency of decreasing was showed even at 72h post operation.In group G and group H,the level of ETX also increased post operation but amplitude of increasing was lower than that of group F(p<0.05).The level of ETX in group H was lower than that of group G at every time point post operation(p<0.05).All groups showed hepatic apoptosis post operation by TUNEL.Group E showed a tendency of decreaing but group F,groupG and group H had the adeverse tendency.3、MAPK signal transduction pathway mediating protection of hepatocyte by SMLS: reverse transcription PCR,Western blotting and TUNEL were used respectively to determine A20 mRNA,A20,P-JNK,P-ERK,P-p38,bcl-2,Bax, caspase-3 and hepatocyte apoptotic rate.Specific inhibitor of MAPK signal transduction pathway SP600125(specific inhibitor of JNK), PD98059(specific inhibitor of ERK)and SB203580(specific inhibitor of p38)were also used in the experiments.The results showed that A20 mRNA and protein level of A20,P-ERK and bcl-2 were obviously higher than those of other groups(p<0.05).But the apoptotic index showed an adverse tendency in which the apoptotic index of group G was marked higher than that of group F(p<0.05).P-JNK,Bax,caspase-3 and apoptotic index of group F were different from those of other groups(p<0.05).Conclusion1、Acute occlusion of portal venous flow in rats with obstructive jaundice can cause mitochondrial energy metabolic block in liver cells.2、The safe tolerant time limit to portal vein ligation in rats with obstructive jaundice and portal flow bypass is sixty minutes.3、Apoptosis of hepatic cells is the mainly representation of ischemia reperfusion injury to liver by portal vein ligation in rats with obstructive jaundice.4、Hepatic apoptosis caused by portal vein ligation in rats with obstructive jaundice is decreased by portal vein perfusion in low temperature with SMLS.5、Acute portal vein ligation can promote hepatic apoptosis by activating JNK signal transduction pathway,mediating the offset balance of bcl-2/bax protein and activating downstream apoptosis-related protein caspase-3.6、The expression of anti-apoptosis protein A20 is activated by hepatic continuous hypothermical perfusion with SMLS,then ERK signal transduction pathway is activated to regulate the balance of bcl-2/bax protein and decrease the expression of apoptosis executive protein caspase-3.更多还原