【作者】 桑新亭；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2007， 博士

【摘要】 第一部分:动物实验——阻断细胞因子的信号传递对大鼠极限肝切除术后存活率的影响及其机制极限肝切除术后的生存是肝外科的一个难题,它引起的爆发性肝衰竭的死亡率在70%～90%,至今虽经种种努力,然效果不显。目前,创伤打击引起的过度炎症反应对机体的损害正越来越被重视。但通过对抗过度炎症反应来保护剩余肝细胞功能的尝试,以及用阿托伐他汀（Atorvastatin）抑制P21-RAS信号在极限肝切除术后的作用,国际、国内尚无报道。目的:本研究用大鼠90%肝切除模型模拟人体极限肝切除,观察Jak-Stat信号通路阻断剂AG490和P21-RAS阻断剂Atorvastatin对大鼠90%肝切除术后一般情况、生存时间和生存率的影响,对肝脏再生和肝功能水平的作用,以及造成以上变化的细胞生物学机制。方法:在标准90%肝切除后,大鼠随机分三组:Ctrl组:术后即时及术后每12小时腹腔内注射生理盐水1ml,直至死亡。Ator组:术前一天至术后三天,每日1次从胃管内注入阿托伐他汀（20mg/kg）。AG490组:术中至术后36小时内每隔12小时腹腔内注射AG490（1mg/kg）。观察一般情况、术后生存时间和生存率、肝脏再生和肝功能水平。并最终检测Jak和Stat信号蛋白磷酸化水平。结果:对照组大鼠在90%肝切除术后24小时的死亡率达100%,病理检查提示术后剩余肝细胞有明显损害。应用AG490后,术后生存率奇迹般地提高到了25%。Atorvastatin组大鼠的术后生存时间也有显著延长,但仍全部死亡。三组大鼠术后生存时间对比为25.6 h和30.6 h vs.10.7 h,p＜0.05。术后48小时内肝脏已出现再生,而存活大于7天的大鼠,再生后的肝脏已超过切除前的1/2。从肝脏再生指数（LRR）和肝细胞PCNA（Proliferating Cell Nuclear Antigen）计数来看,术后72小时肝脏再生活动达到高峰;而术后48小时尽管肝脏再生已经明显,但从生存情况上看仍未见足够代偿意义的再生。AG490组大鼠的术后转氨酶和血糖水平明显优于Ctrl组、更接近正常水平,并在48h后胆红素有明显下降。两组大鼠的血清蛋白水平均有缓慢下降但无差异。结论:（1）肝极限切除术后前48小时肝脏增生不足以代偿其功能,种种有害因子尤其是过度炎症反应损害了剩余肝细胞功能,导致动物死亡,此时需用有效方法来帮助度过此危险期。（2）本研究在国际、国内首先报道了应用细胞因子信号阻断剂AG490在动物模型上显著提高了极限肝切除术后大鼠的生存率,而阻断P21-RAS的Atorvastatin也能延长极限肝切除术后大鼠的生存时间。扩大肝切除术后过度的炎症反应对剩余肝细胞的破坏作用比我们想象的更为严重;本结果所揭示扩大肝切除术后抗过度炎症的新概念为进一步临床实践奠下了一定的理论基础。（3）本研究还揭示了其中的部分机制,研究证实实验组大鼠的肝功能较对照组有明显改善。这种作用主要受益于AG490对剩余肝功能的保护,而非促进肝脏再生。术后早期血糖的改变有敏感的反应,可以成为肝脏手术预后的主要指标之一。第二部分:临床实验——乌司他丁对不同范围肝脏切除影响及其机制的前瞻性临床研究前期的动物实验证实,应用细胞因子信号传递阻制剂AG490,使90%肝切除的大鼠术后生存时间得以明显延长。乌司他丁（Urinary TrypsinInhibitor,UTI）具有对抗炎症等特性,可以保护肝切除术后的肝脏功能;但具体是何种炎症因子起作用、又如何起到保护肝功能的作用,目前国际、国内均无报道。本部分研究利用乌司他丁（UTI）在临床肝切除病例中进一步证实抑制过度炎症反应对改善肝功能的作用,并为其在临床中的实际应用打下基础。目的:观察乌司他丁对不同肝脏切除手术范围、术后不同时间点多种指标变化的影响。本研究通过前瞻、随机、对照临床研究,进一步探讨炎症介质及氧化反应在乌司他丁保护肝脏术后肝功能中所起的作用及其机理。方法:通过前瞻、随机、对照的方法,将我院肝脏外科中心收治的肝切除患者42例分为治疗组和对照组,治疗组分别于手术当天和术后3d静脉应用乌司他丁;全部患者分别观察药物副作用及手术并发症,并在术前及术后抽外周静脉血。其中30例患者检查肝肾功能、凝血机制、凝血因子-Xa,所有患者测定淋巴细胞亚群分类（CD₄、CD₈）、急性反应蛋白（CRP）水平以及和IL-6、肿瘤坏死因子（TNF-α）、一氧化氮合酶（iNOS）的基因表达。结果:应用乌司他丁后,治疗组患者术后12h ALT和AST均有明显下降,如按肝切除的范围进行区分,乌司他丁的作用主要是对手术范围超过联合肝段切除的病例,至术后60h两组肝脏酶谱已无明显差异。乌司他丁对总胆红素的影响较肝酶滞后,表现为在小范围肝切除患者中,术后60h总胆红素有显著下降。乌司他丁对肝脏术后凝血机制的变化无明显作用。应用乌司他丁药物后,治疗组TNF-α和iNOS均较对照组有明显下降（0.053±0.02和0.12±0.04 vs.0.084±0.01和0.21±0.02,p＜0.05）;IL-1的释放高峰被推后;但对CD₄、CD₈和CRP的变化却无明显作用。结论:乌司他丁对大范围肝切除术后肝脏功能的早期恢复起到促进作用,可能通过特异性抑制或调节某些促炎因子的水平来保护肝脏术后的肝功能;这种保护作用还可能同抗氧化作用有关。更多还原

【Abstract】 Chapter1.Animal trail:The effects and mechanism of survival rate by specific inflammatory signal inhibitor in rats following extensive liver resectionExtensive liver resection,if followed by fulminant liver failure,has a high mortality（70%～90%）.It has long been an unsolved issue to find an effective way to protect and promote the function of residual liver to achieve a better survival.This is a first report concerning the effects of specific inflammatory signal inhibitor AG490 and P21-RAS inhibitor atorvastatin in the extensive liver resection（90%partial hepatectomy,90%PHx）.Objective:Rat 90%hepatectomy was used as model for human extensive liver resection.Observe the effects of P21-RAS inhibitor atorvastatin and cytokine signaling inhibitor AG490 upon general situation,survival time,survival rate, liver regeneration,and liver functions.The possible biomolecular mechanism was also under investigation.Methods:Rats were divided randomly into three groups after surgery:Ctrl Group:given normal saline 1ml intraperitoneally,immediately and every 12 hours after the operation,till death occurred;Ator Group:administrated with atorvastatin(20mg·kg^-1·d^-1)through NG tube,one day before and three days after the surgery;AG490 Group:given AG490(1mg·kg^-1·12h^-1)intraperitoneally,immediately and through 36 hours after the operation.The survival situation,liver regeneration,and liver functions of the rats were observed and recorded.Efforts were also made to detect the phosphorylation of signal proteins Jak and Stat.Results:The rats in the Ctrl Group had 100%mortality rate within 24 hours. Severe damage to the residual hepatocytes was observed by pathological examinations.Both atorvastatin and AG490 significantly increased the survival time of rats after surgery（25.6 h & 30.6 h vs.10.7 h,p＜0.05）, especially did the latter.The ratio of survival rate for the Ctrl Group and the AG490 Group was 0%vs.25%.Although atorvastatin increased the survival time,it did not improve the survival rate.None of the rat in the Ator group survived beyond 3 days.During the first 48 hours postoperatively,the remaining liver had started regeneration,which,however,seemed insufficient to compensate its function. Rats who survived up to one week,had regenerated livers heavier than half of their original weight.According to the results of Liver Regeneration Rate （LRR）and hepatocyte Proliferating Cell Nuclear Antigen（PCNA）counting, liver regeneration peaked at the post-operation-time（POT）72h.Although the regeneration was already obvious at POT 48h,little compensatory effects of the regeneration was observed.The AG490 Group had significantly better serum glucose and aminotransferase levels,and serum bilirubin levels decreased rapidly by 48 hours after the operation,when compared with the Control Group.Serum protein levels in both groups decreased slowly,without significance between each other.Conclusions:（1）During the first 48 hours after extensive liver resection,residual liver regeneration is insufficient to compensate for its former liver functions.Death can be resulted from the severe injury to the residual liver caused by various harmful factors,especially the overwhelming inflammatory response.Thus effective measures should be taken to help the residual liver out of this dangerous period of time.（2）It is the first report domestically and internationally to describe that the cytokine signaling inhibitor AG490 significantly increase the survival rate of rats following extensive liver resection.Atorvastatin also significantly prolonged the survival time.The damage towards residual hepatocytes caused by inflammatory response following extensive liver resection is much more serious than we used to consider.The result we obtained implies the theoretical basis of clinical application of certain curations and medications in the future.（3）The partial mechanisms were also studied in this experiment.Rats in the AG490 Group have better liver functions than those in the Control Group, which mainly benefits from protection towards the residual liver,rather than promotion of liver regeneration.The level of blood sugar in early stages is sensitive,which could be important for the prognosis of extensive liver resection.Chapter2.Clinical trail:The effects and mechanism of Ulinastatin following a prospective research in different liver resectionObjective:To investigate the effect of Ulinastatin,a urinary trypsin inhibitor on the outcome of patients undergoing liver resection,including the role and mechanism of inflammatory media and oxidation reaction based on a prospective,randomized,controlled clinical trail.Methods:In this clinical study,42 patients undergoing hepatectomy were randomly divided into Treatment group and Control group.In treatment group Ulinastatin was given on the day of surgery and the three consecutive days. Blood of thirty cases was tested for biochemistry.The levels of CD4,CD8, CRP,and gene expressions of IL-1,Il-6,TNF-alpha,iNOS were measured in blood sample of all patients.Results:Serum ALT and AST levels in treatment group were significantly lower than those in control group 12 hours post-operation,especially in patients undergoing multisegmentectomy.Difference became insignificant at the time point of 60 hours post-operation.Administration of UTI resulted in faster decrease of serum bilirubin level in patients undergoing small volume hepatectomy,and no effect on coagulative function was observed.The TNF-alpha and iNOS levels were significantly lower in the Treatment Group than in the Control Group 12 hours after surgery.The plasma peak of IL-1 was postponed by the administration of UTI.There was no significant change in CD4,CD8,and CRP within these two groups.Conclusions:The protective effects of urinary trypsin inhibitor exist mainly in early stage of major liver resection.The mechanism of protection role of UTI may via the inhibition and regulation of certain pro-inflammatory cytokines,which may also be related to the anti-oxidation effects.更多还原