【作者】 梁四海；

【导师】 张培建； 刘歆农；

【作者基本信息】 扬州大学 ， 外科学， 2007， 硕士

【摘要】 1、目的：木实验在建立大鼠原位肝移植(orthotopic liver transplantation，OLT)模型的基础上，术前对受体大鼠给予一定的低氧预适应(hypoxic preconditioning，HP)，研究低氧预适应对肠道黏膜屏障功能的保护作用，通过血清内毒素定量分析、肠黏膜显微结构的观测得以反映。研究移植肝脏，包括术后2h肝功能、肝组织丙二醛(MDA)和超氧化物歧化酶(SOD)的测定、肝组织中IL-1、TNF-a mRNA表达分析，肝脏超微结构变化。通过与对照组的比较探寻肠道黏膜屏障功能与肝移植肝缺血再灌注早期损伤的关系，从而为临床肝脏移植采取合理的手段减轻肝脏损伤提供理论依据和可能的技术路径。2、方法：2．1 SD大鼠，随机分为3组，A组：原位肝移植组(OLT，n=8)。B组：受体低氧预适应移植组(n=8)，受体大鼠术前24h给予气体流量5L／min的8％氮氧混合气体(含氧气8％，其余为氮气)处理90min。C组：假手术组(n=6)。A、B组均采用Kamada的二袖套法并加技术改进施行大鼠原位肝移植，即经腹主动脉灌肝的快速供肝切取技术。2．2各组大鼠8只均在术后2h处死，经腹主动脉采血作肝功能及血清内毒素检测。切取肝左叶立即置液氮然后放-80℃冰箱保存待脂质过氧化酶及IL-1β、TNF-a的mRNA RT-PCR检测。另取肝脏、回肠组织标本，做切片观测显微和超微结构。剩余12只观察一周存活率。3、结果：(1)每个OLT大鼠手术成功的标准是术后，大鼠即刻苏醒、翻身，并能维持有效的呼吸、心跳。A组和B组有效肝移植大鼠例数各为20，冷缺血时间控制在(51.1±3.2)min，无肝期为(20±2.2)min，两组无明显差异(P＞0.05)。术后一周生存率A组和B组分别为41.7％(5／12)和66.7％(8／12)，有显著意义(P＜0.01)。(2)肝移植大鼠术后2小时，各项指标检测。血清肝功能检测显示：A、B组大鼠ALT、AST与C组相比明显升高，有显著差异(P＜0.01)。B组ALT(231.0±46.1u／L)、AST(684.5±98.4u／L)与A组相比ALT(415±30.2u／L)AST(1217.3±152.0u／L)明显降低，有显著差异(P＜0.01)。B组TBil 2.0±0.5(umol／L)低于A组3.2±0.5(umol／L)，有显著差异(P＜0.01)。肝组织MDA含量与SOD活性的检测：A、B组SOD活性明显低于C组，有显著差异(P＜0.01)。B组SOD活性51.03±5.22(U／mgprot)明显高于A组36.05±4.35(U／mgprot)有显著差异(P＜0.01)。A、B组MDA含量与C组相比差异有显著性(P＜0.01)。B组MDA含量12.34±0.94(U／mgprot)较A组17.92±0.93(U／mgprot)为低，有显著性差异(P＜0.01)。肝组织IL-1 mRNA、TNF-a mRNA表达A组明显高于B组。血清内毒素A组明显高于C组，有显著性差异(P＜0.01)。A组Endotoxin0.86±0.25(EU／L)与B组0.61±0.23相比有显著性差异(P＜0.05)。C组与B组血清内毒素无显著性差异(P＞0.05)。(3)A组、B组肠道黏膜光镜下观察：A组肠道黏膜上皮明显破坏，黏膜下部分腺体结构破坏，腺体间大量炎性细胞浸润(图1A)。B组肠道黏膜上皮局部破坏，黏膜下腺体组织形态尚好，腺体间炎性细胞浸润(图1B)。C组肠道黏膜上皮完整，黏膜下腺体形态正常(图1C)。肝组织透射电子显微镜下观察：A组肝细胞肿胀，内皮细胞变形，线粒体肿胀，呈气球样改变，基质加深、减少，呈絮状改变，嵴减少，排列紊乱，细胞核内染色体核边聚(图2A)。B组肝细胞线粒体肿胀较轻，嵴减少(图2B)。C组细胞形态基本正常，细胞器无明显水肿、变性，线粒体排列正常，无明显肿胀(图2C)4、结论：低氧预适应对受体大鼠无肝期肠道淤血性缺氧诱导的肠道黏膜损伤有保护作用，对移植肝脏早期缺血再灌注损伤具有保护作用。其机制可能与低氧预适应减轻肠道缺氧再氧和损伤，保护肠道黏膜屏障功能有关，减少肠道内菌群异位，减轻毒素对移植肝脏的损伤从而提高移植肝脏的功能。对肠道黏膜屏障的保护减轻移植肝脏缺血再灌注损伤可能为降低术后移植肝失功能提供一条全新的途径。更多还原

【Abstract】 【Objective】: This investigation builds on rats orthotopic liver transplantation(OLT) model. Acceptors administrate hypoxic preconditioning in preoperative 24h.Aim to investigate protection of hypoxic preconditioning on intestinal barrierfunction via quantitative analysis of serum endotoxin and observation of intestinalmucosa microstructure and ultrastructure. To compare liver transplantedpostoperative 2h both B group and A group including serum liver function,analysis of hepatic tissue MDA,SOD, IL-1and TNF-a mRNA expression and thechange of microstructure and ultrastructure.To find the relationship of intestinalmucosa barrier function and liver transplanted early-stage ischemia reperfusioninjury in order to provide a potential approach for clinic hepatic transplantation.【Method】: Adult male Sprague-Dawley (SD) rats were randomly divided into threegroups. A: OLT group (n-8), B: AHP (acceptor hypoxic preconditioning)--OLTgroup (n=8), C: Shame operation group(n=6). Group B, Acceptors were treatedwith 8% nitrogen oxygen atmosphere for 90 minutes by 5L/min flow rate inpreoperative 24hours. Rats orthotopic liver transplantation are according toKamada two-sleeve approach. The sham group was subjected to the surgicalprocedures without liver manipulation and maintained under anesthesia for thesame time. Two hours after the reperfusion, all animals underwent relaparotomythrough the previous incision. The abdominal aorta was punctured and bloodsamples were collected for measurement of plasma endotoxin levels, liverenzymes and serum total bilirubin (TBil). liver tissue samples from the left lobewere collected, washed with cold physiologic saline solution and immediatelyfrozen in -80℃refrigerator until malondialdehyde (MDA) levels, superoxide dismutase (SOD), IL-1βand TNF-a mRNA RT-PCR determination. Liver tissuesamples and ileal mucosal samples were biopsied, fixed in 2.5% glutaraldehydeand 10% formaldehyde for later histological study by electronic-microscopy andoptic-microscopy respectively. Twelve rats were left to observe the one weeksurvival rate.【Results】:1. The standard of stable OLT models is able to maintain steady respiration andheartbeat, revive and turn body over post-operation. Cold ischemic time,unhepatic period of OLT were controlled within 50±3.15min,20±2.15minrespectively. The difference of the operation and the conservation of the graftcould be neglected. The totle number of animal treated with orthotropic livertransplantation in group A and group B respectively is 20, of which 8 wasrandomly selected to obtain the samples with 12 left to observe the survival.The one week survival rate of group B,41.7% (5/12), was significantly longerthan group A—66.7% (8/12) (P＜0.01)..2. The ALT and AST in A group and B group is significantly higher than that ingroup C, P＜0.01. The ALT, AST and TBIL were higher than that in B group aswell, P＜0.01. No significance is exist about TBIL between A group and B group,(P＞0.05). Liver MDA concentration in C group was significantly lower than that ingroup A and groupB, but the level of liver SOD activity was reversely(P＜0.01).Liver SOD activity, 51.03±5.22 (U/mgprot),in group B was higher than that ingroup A, 36.05±4.35(U/mgprot),P＜0.01. Liver MDA concentration in group A washigher than that in group B as well. The expression of liver IL-1 mRNA andTNF-a mRNA in group A was significantly higher than B,(P＜0.01).Plasmaendotoxin levels in A group,0.86±0.25 (EU/L), was significantly higher than Cgroup(P＜0.01) and B group,0.61±0.23(EU/L), (P＜0.05). No significance isexist compared endotoxin levels in B group with that in C group, (P＞0.05). 3. Transmission electron microscopy revealed that the ultra micro-construction ofthe liver graft in C group, but in A group the liver graft presented hepatic celledema, degeneration even necrosis, sinus stegnosis. The lesion of the hepaticcell and mesenchyme was milder in group B than that in group A. Opticsmicroscopy revealed that the micro-construction of ileal mucosa in shamegroup stayed normal. The epithelium of ileal mucosa in group A were lost anddisrupted. The lesion of the epithelial basement membrane in group B wasmilder than that in group A.【conclusion】This investigation implied that recipient hypoxic preconditioning may relieve ratintestinal mucosa injury induced by intestinal stagnant anoxia in anhepatic phase and liverearly-stage ischemia reperfusion injury during liver transplantation course. Hypoxicpreconditioning mitigated hypoxia/reoxygenation-induced intestinal injury and protectintestinal mucosa barrier function,which alleviated intestinal bacterium translocation and theendotoxin absorbed into systemic circulation so that it can improve the graft liver function andprolong the graft survival.Hypoxic preconditioning on recipient may provide a novel approachto protecting the reperfusion injury of liver transplanted and decreasing the rate of liver nofunction in postoperation.更多还原