兔肝部分切除术后门—腔分流对肝再生影响的实验研究

发布时间：2018-02-27 07:13

  本文关键词： 兔 肝脏解剖 SFSS 动物模型 门静脉压力 门-腔分流 肝再生　出处：《昆明医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景 小肝综合征(small-for-size syndrome, SFSS)是发生在扩大肝切除或部分原位肝移植后由于肝脏体积过小导致功能上不能满足受体的代谢需求而出现的一种临床综合征。SFSS最常见于成人—成人部分原位活体肝移植,供肝体积较小时可能导致受体门静脉血流高灌注和持续性门静脉高压,由此引起一系列严重的病理生理紊乱。其临床表现为：术后持续性黄疸、凝血功能障碍、门静脉高压、顽固性腹水,严重者进一步诱发脓毒血症、胃肠道出血等并发症。 SFSS的病因及发病机制至今尚未完全清楚,目前普遍认为主要是由于功能性肝脏体积过小,从而引起持续性门静脉高压,持续性的门静脉高灌注进一步导致肝窦内皮细胞损伤继而造成肝实质细胞的损伤,影响肝细胞再生,最终导致肝功能不全。除此之外,还可能与其他多种因素有关,如移植物的大小、潜在病变、肝再生能力、流入道和流出道情况,受者病情轻重等等。正因为如此,SFSS的防治措施也一直在不断的积极探索之中。 目前预防SFSS的策略主要集中在以下3个方面[6]:(1)增加肝脏移植物的体积；(2)控制门静脉的压力；(3)改进肝脏静脉回流的方式。大部分学者普遍认同门静脉高灌注和门静脉高压对小体积肝脏移植物有破坏作用,因此降低门静脉灌流和压力也成为预防SFSS的主要手段。而目前常用的手术方式包括脾动脉结扎术和门-体分流术。 脾动脉结扎、栓塞操作简单,创伤小,对门静脉灌注压力的调节作用有限,可并发术后门静脉血栓和腹腔感染；门静脉分流能有效调节门静脉的灌注压,近年来,一些研究者采用门静脉-腔静脉分流来防治SFSS的发生。但是,有学者发现门-腔静脉分流在活体肝移植术后数周内虽能有效减少SFSS的发生,并能提高移植肝的存活率,后期却出现移植物逐渐萎缩、肝再生受到抑制的现象。 由此看来,在扩大肝切除或部分肝移植术后维持适当的门静脉灌注压在余肝再生过程中发挥了重要作用。本实验拟建立SFSS实验动物模型,并实施门-腔分流调控门静脉的灌注血流,观察门静脉灌注压对肝再生的影响。 目的 1.对兔肝脏分叶及附属管道进行应用解剖学研究,并在此基础上建立兔SFSS动物模型。 2.在SFSS动物模型的基础上实施门-腔分流,观察门静脉灌注压力的变化对肝功能和肝再生的影响。方法 1.兔肝脏及其附属管道应用解剖学研究：选择由昆明医科大学实验动物中心提供的健康成年日本大耳兔20只,体重范围1.5kg-2.5kg。经耳缘静脉按照1ml/kg注射3%戊巴比妥钠麻醉。固定其四肢,腹部褪毛。(1)肝脏在体与离体解剖学观察：取正中切口进入腹腔,离断肝周韧带,充分暴露肝脏并对其进行在体解剖学观察。处死动物后,经门静脉灌注生理盐水冲洗血管。完整取下肝脏对其进行离体解剖学观察。(2)全肝及各肝叶称重,并计算各肝叶占全肝脏及兔体重百分比。(3)制作门静脉和肝静脉管道灌注标本。经肠系膜上静脉插管结扎其尾侧端,注入管道铸型剂(自凝造牙粉和自凝牙托水按10g：10ml混合)制作门静脉管道铸型标本。结扎肝后腔静脉头侧、尾侧端,同上注射管道铸型剂制作肝静脉管道铸型标本。待管道铸型标本硬固后,取出肝脏。将其放置于20%氢氧化钠溶液中浸泡腐蚀显露管道标本。SPSS13.0软件对数据进行描述性分析,计量数据结果以“均数±标准差(x±s)”表示。 2. SFSS动物模型的建立：将42只兔随机分为5组,分别为假手术组(n=2),A组(n=10)为切除“左中叶+右中叶”组,B组(n=10)为切除“左外叶+左中叶+右中叶”组,C组(n=10)为切除“尾状叶+左中叶+右中叶+右外叶”组,D组(n=10)为切除“左外叶+左中叶+右中叶+右外叶”组。经耳缘静脉按照1ml/kg注射3%戊巴比妥钠麻醉。固定四肢,腹部褪毛,碘伏消毒,铺无菌单。取腹部正中切口进入腹腔。应用无血切肝技术,分别对A组、B组、C组及D组实施肝叶切除术。观察术后兔活动及进食情况。术前和术后存活兔分别于第1、3、5、7天麻醉后经耳缘静脉采血留取标本检测ALT、TB、PT,然后开腹获取肝组织标本行组织病理学检查。术后死亡兔行肝脏剖检。计量数据结果以“均数±标准差(x±s)”表示,应用SPSS13.0统计软件对数据进行F检验和x2检验,检验水准a=0.05。 3.在SFSS的基础上实施门-腔分流,调控门静脉灌注压力,观察肝再生：将120只兔随机分为3组,分别为E组(n=40)为“SFSS模型”组,F组(n=40)为‘'SFSS基础上的门-腔分流”组,G组(n=40)为“门-腔分流调控”组。同样经耳缘静脉以1ml/kg注射3%戊巴比妥钠麻醉,之后固定四肢,腹部褪毛,碘伏消毒,铺无菌单。取腹部正中切口进入腹腔。应用无血切肝技术,分别对E组、F组及G组实施“左外叶+左中叶+右中叶”的肝叶切除术,同时对F组、G组进一步行改良后的门-腔静脉分流术。观察术后兔生存情况。术前和术后E组、F组存活兔分别于第1、3、5、7、9、11、13、15、17、19天再次开腹测量门静脉压力值并对余肝进行称重观察肝再生。而G组存活兔则统一于术后第7天开腹结扎封闭分流口后分别于第9、11、13、15、17、19天开腹测量门静脉压力值及余肝重量进一步观察调控门-腔分流后的门静脉压力值变化对肝再生的影响。计量数据结果以“均数±标准差(x±s)”表示,应用SPSS13.0统计软件对生存率组间比较行Log Rank检验,组间差异采用重复测量方差分析,以P0.05有统计学意义。 结果 1.兔肝肝裂明显,依据肝叶形态、肝裂走形和门静脉主干分支形式将兔肝脏分为五叶：尾状叶、左外叶、左中叶、右中叶和右外叶。全肝质量为(60.13±16.11)g,占其质量百分比为(2.88±0.06)%,各肝叶质量分别为(3.93±1.13)g、(15.93±3.50)g、(14.83±3.31) g、(15.08±4.34) g、(12.08±3.55) g占全肝质量百分比分别为(6.22±1.02)%、(25.44±2.55)%、(23.72±2.71)%、(24.15±5.21)%、(19.32±4.23)%。左中叶和右中叶根部肝组织融合,其余各肝叶相对独立,尾状叶包括相对独立的乳头突和尾状突两部分。各肝叶有相对独立的Glisson系统且肝静脉走行于肝蒂内 2.根据上述测得的各肝叶质量及不同肝叶组合占全肝质量百分比数据,A、B、C、D四组保留肝叶占全肝质量百分比分别约为50%、28%、25%和6%,术后一周存活率分别为100%、50%、20%和0%。术后A组与假手术组比较,ALT、TB、PT数值变化不明显(P0.05)；B、C两组ALT、TB、PT在术后第1天明显升高,第3天达到高峰,随后逐渐降低,至第7天仍较对照组和A组增高(P0.05)；而B、C两组数值术后第1、3、5、7天组间比较,C组虽较B组有所升高,但两者差值较小(P0.05)。剖检术后7d内自然死亡的兔肝,可见不同程度的淤血、淤胆、出血,余肝肿胀、坏死,胃肠道淤血,浑浊腹水；相比之下,存活7d者则可见肝体积明显增大,与周围组织致密粘连,边缘圆钝,似圆盘状,腹腔内少量清亮腹水。肝组织病检可见：A组汇管区无明显有丝分裂象和多倍体细胞；B、C两组肝组织淤血水肿明显,可见中央小静脉及肝窦扩张,汇管区中性粒细胞浸润明显,毛细血管内胆栓形成,肝实质可见点状坏死,肝细胞气球样变和脂肪变性,随着时间延长,肝实质细胞增生逐渐活跃,并逐步出现多倍体细胞,但肝实质细胞增生比A组延迟。 3.根据上述实验结果,选择实验B组建立较稳定的SFSS动物模型,并在其基础上实施门-腔分流手术。术后19天E、F、G三组存活率分别为10%、30%和60%。术后E组表现为典型的SFSS特点,余肝组织充血水肿,肝实质破坏较多,肝再生表现不明显,并且随着时间的延长,余肝逐渐出现不能程度的坏死、胃肠道淤血等情况,实验动物相继死亡,最后仅4只存活时间达到19天。而F、G两组因在扩大肝切除的基础上实施门-腔分流手术,术后短时间内降低了门静脉的灌注压力,从而减少了过高的门静脉灌注血流量对余肝组织的损坏作用,余肝组织充血水肿程度较轻,术后动物死亡率均较E组明显下降,生存时间有所延长,并表现出不同程度的肝脏再生。至术后7天,因F组的门静脉压力持续降低,对余肝再生的机体代谢支持逐渐减少,从而导致肝脏再生减缓,肝功能逐渐恶化,则继续出现动物死亡现象,最后有12只存活时间达到19天。因此,G组在术后第7天又再次开腹结扎门-腔分流口后,门静脉灌注压力再次逐渐升高,从而满足了余肝持续再生的机体代谢需求,动物死亡率较F组进一步下降,肝脏再生明显,最后有24只存活时间达到19天。 结论 1.兔肝脏解剖学既与犬、鼠、猪等哺乳类动物类似,又具有其自身的特点。本实验通过对兔肝脏在体、离体解剖学和肝脏管道灌注标本的观察,规范了文献中对兔肝解剖学的混乱描述。并在此基础上,通过测量全肝及各肝叶质量,计算出各肝叶所占全肝质量百分比,为在该方面的基础研究提供数据支持。 2.根据兔肝解剖学特点行不同质量百分比肝叶切除术制作SFSS模型,当保留右外叶和手术切除相对困难的尾状叶后,即余肝占全肝质量百分比约为28%时,术后存活率较高并可表现出典型的SFSS。而且兔体积适中,围手术期管理和手术操作相对简便,可作为研究SFSS发病机制及防治措施较为理想的实验动物模型。 3.门-腔分流术后可明显降低门静脉灌注压,促进肝再生,有效延长术后动物生存时间；过度分流,门静脉灌注压低于正常值则肝再生受到抑制。在扩大肝切除术后发生SFSS情况下,采用门-腔分流手术维持适度增高的门静脉灌注压可明显促进肝再生。
[Abstract]:background
Small liver syndrome (small-for-size, syndrome, SFSS) occurs in hepatic resection or partial orthotopic liver transplantation after due to liver volume is too small to function can not meet the demand of the metabolic receptor a clinical syndrome.SFSS is most common in adult adult living donor liver transplantation for partial orthotopic liver volume is small, may lead to the receptor of portal vein blood flow perfusion and high persistence of portal hypertension, which caused a series of serious pathophysiological disorder. Its clinical manifestations include persistent postoperative jaundice, coagulopathy, portal hypertension, refractory ascites, which further induce sepsis, gastrointestinal bleeding and other complications.
The etiology and pathogenesis of SFSS has not yet entirely clear, the prevailing view was mainly due to functional liver volume is too small, resulting in persistent portal hypertension, persistent portal hyperperfusion and further lead to injury of sinusoidal endothelial cells and caused liver parenchyma cell damage, liver cell regeneration, eventually lead to liver function. In addition, it may be related to other factors, such as graft size, potential lesions, liver regeneration, inflow and outflow tract, the severity and so on. Because of this, SFSS control measures have also been active in exploring.
At present, SFSS prevention strategies mainly focus on the following 3 aspects: (1) [6] increased liver graft volume; (2) control of portal vein pressure; (3) improvement of hepatic venous return way. Most scholars generally agree on portal vein perfusion and portal hypertension have a destructive effect on the small volume of liver graft plants, therefore reduce portal vein pressure and perfusion has become the main means of preventing SFSS. And the current commonly used surgical methods including splenic artery ligation and portosystemic shunt.
Ligation of splenic artery embolization, simple operation, small trauma, portal vein perfusion pressure regulation is limited, can be complicated with postoperative portal vein thrombosis and infection of abdominal cavity; portal vein shunt can effectively regulate the portal vein perfusion pressure, in recent years, some researchers use the portal vein to vena cava shunt occurred to the prevention and treatment of SFSS. However, there are researchers found that portacaval shunt in living donor liver transplantation after a few weeks can effectively reduce the incidence of SFSS, and can improve the survival rate of liver transplantation, but late graft gradually atrophic, inhibited the phenomenon of liver regeneration.
Therefore, to maintain the proper pressure in the portal vein perfusion during liver regeneration than plays an important role in hepatic resection or liver transplantation. This study was to establish the animal model of SFSS, and the implementation of perfusion portocaval shunt regulation of portal vein, to observe the effect of portal vein perfusion pressure on liver regeneration.
objective
1. the applied anatomy of the rabbit's liver lobules and accessory pipes was carried out, and a rabbit SFSS animal model was established on the basis of this.
2. the portal cavity shunt was carried out on the basis of the SFSS animal model, and the effects of the changes of portal vein perfusion pressure on liver function and liver regeneration were observed.
Study on applied anatomy of 1. rabbit liver and its affiliated pipeline: from the experimental animal center of Kunming Medical University health 20 adult Japanese white rabbits, weight 1.5kg-2.5kg. intravenously with 1ml/kg injection of 3% pentobarbital anesthesia. The fixation of limbs, abdominal faded hair. (1) the liver in vivo and in vitro anatomy observation: the median incision into the abdominal cavity, severed ligaments around the liver, liver and fully exposed were observed in vivo anatomy on it. Kill the animal, via the portal vein infusion of saline irrigation vessels. The complete pathological anatomy observation in vitro. (2) and the total hepatic lobe of the liver and liver and weighing, calculation the rabbit liver weight percentage. Ye Zhanquan (3) made of portal vein and hepatic vein perfusion specimens. After ligation of the superior mesenteric vein intubation caudal, injection pipe casting agents (self curing polymer and self curing denture water by 10g:10ml Mixed) making cast specimens of portal vein ligation of hepatic vena cava pipeline. Cephalic, caudal, ditto injection pipe casting agents making cast specimens of hepatic vein duct. To cast specimens of hardened pipe, remove the liver. Placed in 20% sodium hydroxide solution immersion exposure pipe specimens.SPSS13.0 for descriptive analysis of data the results of measurement data, the standard deviation (x + s) ".
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