三氧化二砷对逆行隔离灌注大鼠肝脏毒性的观察

目的 观察不同剂量三氧化二砷(As2O3)在大鼠逆行隔离灌注(RIHP)模型中对肝脏毒性作用.方法 104只体重300～400 g雄性SD大鼠中8只为术前正常对照组,其余大鼠分A、B、C、D 4组,每组24只.A组灌注不含As2O3的乳酸林格氏液,B、C、D组灌注As2O3的剂量分别为0.75 mg/kg、1.5 mg/kg、3 mg/kg.实验组均采用RIHP,即经肝动脉灌注As2O3溶液,经肝静脉逆行灌注乳酸林格氏液,门静脉为流出道;灌注时间为15 min,速度1 ml/min.对照观察实验组术后1、2、37 d各时点肝酶学变化、肝组织学改变和术后生存情况;检测C组大鼠术中和术后第1天的肝循环和体循环中As2O3浓度.结果 实验组ALT、AST峰值均出现在术后第1天,至术后3～7 d恢复正常.A和B组ALT、AST峰值间差异无统计学意义,A和C,A和D,B和C,B和D、C和D各组间ALT、AST峰值差异均有统计学意义(FALT=40.811,FAST=48.212,均P<0.01).D组术后第7天时仍与术前正常对照组及A、B、C各组间差异均有统计学意义(FALT=13.928,FAST=17.942,均P<0.01),且术后肝组织出现片状坏死等较严重的损害.肝循环和体循环As2O3血药浓度峰值分别为(13.21±0.82)μg/ml和(0.09±0.008)μg/ml,二者比较差异有统计学意义(t=35.758,P<0.01).结论 在人鼠RIHP模型中,当As2O3灌注剂量不超过1.5 mg/kg时肝脏遭受的损害是可逆的.     

HTK液逆行灌注可减轻肝脏隔离化疗对大鼠的损伤

目的:建立大鼠肝脏隔离灌注化疗模型;探讨在大剂量化疗药物肝脏隔离灌注过程中,HTK液经肝静脉逆行灌注对肝脏的保护及减少全身泄漏作用。方法:将75只体重300～350g的雄性SD大鼠随机分为3组,每组25只;手术建立大鼠隔离灌注化疗模型。A组经肝动脉灌注含30mg/L三氧化二砷的林格液,门静脉灌注林格液,肝静脉为流出道。B组经肝静脉灌注林格液,门静脉为流出道,余同A组。C组经肝静脉灌注4℃组氨酸-色氨酸-酮戊二酸(Histidine-Tryptophan-Ketoglutarat,HTK)液,余同B组。各组于术后1、2、3、7d各随机处死大鼠5只,进行血清肝功能及肝组织病理学检查。A组和B组大鼠术中检测体循环和肝循环中的血药浓度。结果:各组动物血清ALT和AST峰值均出现在术后第1天,其后开始下降,至术后7d恢复正常。术后第1、2、3d,3组间的血清ALT、AST均数均有显著性差异(P0.05);光镜下观察肝组织,进一步证实了上述结果。A组与B组间体循环血药浓度差异有统计学意义(P0.05),而两组间的肝循环血药浓度相类似;表明逆行隔离灌注的体循环泄漏率较顺行隔离灌注组为低。结论:低温HTK液逆行灌注可显著减轻肝脏隔离化疗对大鼠的损伤,提高手术的安全性。     

区域隔离肝脏灌注的研究

本研究旨在探讨区域隔离肝脏灌注(RIHP)的可行性 ,并初步评价其隔离效果 ,化疗药物局部积聚效果及肝脏损害情况。一、材料与方法1.动物选择 :幼猪 2 1头 ,随机分为 3组 :经动脉组 (实验A组 ) 7头 ;经动脉和门静脉联合组 (实验B组 ) 7头 ;对照组(C组 ,经肝固有动脉灌注化疗组 ) 7头。3组动物的体重差异无显著性 (P >0 .0 5 )。2 .手术操作 :麻醉后上腹部屋顶形切口进腹 ;A组游离左肝动脉 ,近心端结扎 ,远心端插管 ;解剖出左肝静脉 ,远端阻断 ,近端插管。B组则在左肝动脉插管后 ,继续解剖游离出左门静脉 ,近端阻断 ,远端插管 ,左肝静脉处…     

大鼠原位肝脏低温灌注和复流模型的建立及意义

目的建立一种大鼠肝脏原位低温灌注再复流模型,用于离体及活体供肝肝切除术的研究。方法大鼠78只,分成4组,A、B组分别用乳酸林格氏液和自制灌注液灌注,C组仅行全肝血流阻断,D组作正常对照。脾静脉和右肾上腺静脉分别作为灌注液流入和流出道,行在体原位低温灌注,观察再复流后对肝细胞和肝窦内皮细胞的影响。结果C组再复流2h和6h血清透明质酸浓度为（250.0±24.1）μg/L、（290.4±37.0）μg/L,分别高于A组（142.9±30.1）μg/L、（200.0±17.5）μg/L,差异均有非常显著性（P<0.01）;B组浓度为（63.3±16.2）μg/L、（62.1±14.6）μg/L,与A组相比浓度更低（P<0.01）。结论该模型方法简单,易成功,可用于离体和活体供肝肝切除防止肝脏损伤的研究。     

逆行隔离灌注化疗可减轻对大鼠肝脏的毒性及药物的泄漏

Objective The retrograde isolated hepatic perfusion (RIHP) model was used to compare with the isolated hepatic perfusion (IHP) model in reducing the rate of normal hepatic tissue toxicity and peripheral drug leakage during chemotherapy in rat liver. Methods A total of 90 male Sprague-Dawley rats weighing 300-350 g were randomized into 3 groups with 30 rats in each. Group A: perfusion with Lactated Ringer'S Solution through arteria hepatica (RA) and portal vein (PV),the inferior vena cava was used as an outflow tract of perfusate. Group B: For isolated hepatic perfusion (IHP), Fluorouracil (5-FU) was added into the perfusate at a dose of 350mg/kg and introduced in to the liver through arteria hepatica, portal vein was perfused by Lactated Ringer'S Solution, and the inferior vena cava was used as an outflow tract of perfusate. Group C: by using retrograde isolated hepatic perfusion (RIHP), the solution which contains 350 mg/kg Fluorouracil (5-FU) was also introduced through arteria hepatica, the inferior vena cava was introduced with Lactated Ringer'S Solution;the portal vein was used as an outflow tract of the perfusate. On day 1, 3, 5 and 7 after the perfusion in all groups, blood serum ALT test and liver histopathology test were performed. The peripheral blood drug levels were measured with high performance liquid chromatographic(HPLC) system in group B and group C. Results The survival rate was 90%, 86.7% and 90% in group A, B and C,respectively. No statistically significant difference was observed in the survival rate among the 3groups. In all the three groups, serum ALT levels were the highest on the first day after IHP: (481.6±207.6)μmol/LingroupA;(1641. 6±658.0) μmol/LingroupBand( 913. 0±353. 5)μmol/Lin group C. Significant higher serum ALT levels were observed by comparing group B and C with A(P＜0. 05). Meanwhile, the serum ALT levels were significantly higher in group B than in group C (P＜0.05). The peaks of peripheral blood drug concentration during the perfusion were 131.2±29.4μg/ml in group B and 65.3±28. 4μg/ml in group C. Significant difference was observed (P＜0. 05). Liver biopsies of group A showed mild changes on the first day after IHP and returned to normal after 7 days. Group B showed severe changes on the first day after IHP and local necrosis still existed after 7 days. Group C showed moderate changes as compared with group B on the first day after IHP and also returned to normal after 7 days. Conclusion Retrograde isolated hepatic perfusion (RIHP) can reduce the liver toxicity compared to isolated hepatic perfusion (IHP). Hopefully, RIHP will be considered as a safer way in regional chemotherapy in liver cancer.     

逆行隔离灌注化疗可减轻对大鼠肝脏的毒性及药物的泄漏

Objective The retrograde isolated hepatic perfusion (RIHP) model was used to compare with the isolated hepatic perfusion (IHP) model in reducing the rate of normal hepatic tissue toxicity and peripheral drug leakage during chemotherapy in rat liver. Methods A total of 90 male Sprague-Dawley rats weighing 300-350 g were randomized into 3 groups with 30 rats in each. Group A: perfusion with Lactated Ringer'S Solution through arteria hepatica (RA) and portal vein (PV),the inferior vena cava was used as an outflow tract of perfusate. Group B: For isolated hepatic perfusion (IHP), Fluorouracil (5-FU) was added into the perfusate at a dose of 350mg/kg and introduced in to the liver through arteria hepatica, portal vein was perfused by Lactated Ringer'S Solution, and the inferior vena cava was used as an outflow tract of perfusate. Group C: by using retrograde isolated hepatic perfusion (RIHP), the solution which contains 350 mg/kg Fluorouracil (5-FU) was also introduced through arteria hepatica, the inferior vena cava was introduced with Lactated Ringer'S Solution;the portal vein was used as an outflow tract of the perfusate. On day 1, 3, 5 and 7 after the perfusion in all groups, blood serum ALT test and liver histopathology test were performed. The peripheral blood drug levels were measured with high performance liquid chromatographic(HPLC) system in group B and group C. Results The survival rate was 90%, 86.7% and 90% in group A, B and C,respectively. No statistically significant difference was observed in the survival rate among the 3groups. In all the three groups, serum ALT levels were the highest on the first day after IHP: (481.6±207.6)μmol/LingroupA;(1641. 6±658.0) μmol/LingroupBand( 913. 0±353. 5)μmol/Lin group C. Significant higher serum ALT levels were observed by comparing group B and C with A(P＜0. 05). Meanwhile, the serum ALT levels were significantly higher in group B than in group C (P＜0.05). The peaks of peripheral blood drug concentration during the perfusion were 131.2±29.4μg/ml in group B and 65.3±28. 4μg/ml in group C. Significant difference was observed (P＜0. 05). Liver biopsies of group A showed mild changes on the first day after IHP and returned to normal after 7 days. Group B showed severe changes on the first day after IHP and local necrosis still existed after 7 days. Group C showed moderate changes as compared with group B on the first day after IHP and also returned to normal after 7 days. Conclusion Retrograde isolated hepatic perfusion (RIHP) can reduce the liver toxicity compared to isolated hepatic perfusion (IHP). Hopefully, RIHP will be considered as a safer way in regional chemotherapy in liver cancer.     

FK506对肝脏保存再灌注中ICAM-1 mRNA表达的影响

本研究建立离体大鼠肝脏保存再灌注模型 ,观察肝脏保存再灌注中细胞间粘附分子 1(ＩＣＡＭ 1)ｍＲＮＡ表达变化和ＦＫ5 0 6的作用。材料和方法一、动物模型及分组健康Ｗｉｓｔａｒ雄性大鼠 5 4只 ,体重 2 0 0～ 2 5 0ｇ ,随机分为9组 ,每组 6只。于下腔静脉注入肝素 5 0Ｕ ,游离门静脉 ,胆总管 ,肝上、下腔静脉 ,分别插管 ,右肾静脉平面结扎 ,切断肝下下腔静脉 ,经门静脉原位灌注 1℃乳酸林格液 2 0ｍｌ,迅速切除肝脏。正常组 :(保存 0ｈ组 ,6只 )切除肝脏后立即与灌注系统连接 ,行离体鼠肝循环再灌注 ,灌注液为自制ＫＨＢ平衡液 ,加入新鲜…     

经门静脉原位灌注自制肝保护液对梗阻性黄疸大鼠门静脉阻断期间肝脏的保护作用

目的:研究梗阻性黄疸大鼠门静脉阻断期间给予低温灌注自制肝保护液对肝脏的保护作用.方法:72只大鼠行胆总管结扎1周后随机均分为对照组(胆道再通,仅游离门静脉);门静脉阻断(PVO)组(胆道再通后,行PV0 1.5 h);乳酸林格液灌注组(胆道再通,PVO过程中将4℃乳酸林格液注入门静脉肝侧);自制肝保护液灌注组(胆道再通,PVO过程中将4℃自制肝保护液注入门静脉肝侧).于术后0,6,24 h采集肝组织标本,采用Western blot法检测肝组织bcl-2,bax及caspase-3蛋白的表达,TUNEL法检测肝细胞凋亡情况.结果:与对照组比较,PVO组术后个时间点肝组织bcl-2蛋白略有增加,但差异无统计学意义(均P＞0.05),而2个灌注组肝组织bcl-2蛋白表达在术后6,24 h均明显升高(均P＜0.05),且自制肝保护液灌注组升高更为明显(均P＜0.05);PVO组术后6,24 h肝组织Bax,easpase-3蛋白表达明显升高及肝细胞凋亡率明显增加(均P＜0.05),两种灌注液均能明显抑制Bax和easpase-3蛋白表达的升高和肝细胞凋亡率的增加(均P＜0.05),且自制肝保护液的以上作用更为明显(均P＜0.05).结论:自制肝保护液对合并梗阻性黄疸的PVO具有肝保护作用,其机制与抑制线粒体途径的肝细胞凋亡有关.     

门静脉减压术和FK506对扩大肝切除术影响的实验研究

目的阐明扩大肝切除术后门静脉减压术和FK5 0 6的有效性。方法将实验猪随机分成 4组 ,每组 10头。A组为切除 80 %肝脏加门静脉腔静脉分流术加术前FK5 0 6处理 ,B组为切除80 %肝脏加门静脉腔静脉分流术 ,C组为切除 80 %肝脏加FK5 0 6 ,D组为对照组 (仅切除 80 %肝脏 )。观察血流动力学、肝功能和剩余肝组织微循环变化。结果A组和B组的 5d生存率分别为 80 %和6 0 % ,C组和D组为 30 %和 2 0 % (P <0 0 5 )。术后PVP≤ 2 0 0mmH2 O时生存率为 90 % (17/19) ,PVP >2 0 0mmH2 O时生存率为 10 % (P <0 0 1)。肝切除后 12h时A组和B组ALT低于C组和D组 (P <0 0 1)。术后 1d时A组剩余肝组织的微循环最好 (P <0 0 5 )。结论猪 80 %肝切除加门静脉腔静脉分流及FK5 0 6预处理能提高术后生存和促进剩余肝组织再生 ,PVP可作为判断扩大肝切除术预后的一个较好的指标。     

肝动脉灌注和外周静脉输注5-FU在大鼠肝脏和血液中的浓度分布的实验研究

目的探讨经肝动脉区域灌注和外周静脉注射5-FU在大鼠血液和肝脏组织中的浓度分布规律及差异。方法本研究将24只Wistar大鼠随机分为两组：A组为肝动脉灌注组;B组为外周静脉（颈静脉）输注组,分别经肝动脉插管区域灌注与经颈静脉注射5-FU,剂量为20mg/kg体重,每组均有6只大鼠取血,6只取肝组织样本。采用高效液相色谱法测定血浆及肝脏组织中5-FU的含量,并计算5-FU在肝脏和血浆中的药动学参数、穿透比率、穿透指数和相对治疗优势度。结果经外周静脉注射后肝脏组织的药物峰浓度（Cmax）和药物时量曲线下面积（AUC）分别为13.79±4.56μg/g,342.20±108.20μg·h/g;血浆Cmax和AUC分别为36.85±5.96μg/ml,842.00±158.00μg·h/ml。肝动脉灌注5-FU后肝脏组织药物Cmax和AUC分别为29.58±4.30μg/g,794.60±115.40μg·h/g;血浆Cmax和AUC分别为24.39±4.63μg/ml,639.70±133.80μg·h/ml。结论与外周静脉注射全身化疗比较,区域性肝动脉灌注5-FU可显著提高肝脏组织中的药物浓度,同时减少化疗药物在外周血中的分布。     

True versus Mild Hyperthermia during Isolated Hepatic Perfusion: Effects on Melphalan Pharmacokinetics and Liver Function

Hyperthermic antiblastic isolated hepatic perfusion (IHP) with melphalan has been recently proposed as an alternative therapeutic option for patients with unresectable liver tumors. Although melphalan–heat antiblastic synergism is at a maximum at temperatures higher than 41 °C, IHP has so far been performed in humans at lower temperatures. In this experimental work, we compared IHP under mild versus true hyperthermic conditions in terms of drug pharmacokinetics and liver function. Ten pigs were submitted to IHP with melphalan 1.5 mg/kg at a mean temperature of 40 °C (group A, n = 5) or 42 °C (group B, n = 5). After a 60-minute perfusion, a 15-minute washout was performed. Perfusate-to-plasma leakage was monitored using scintigraphy. Throughout perfusion, samples from the systemic blood, perfusate, and liver parenchyma were obtained to measure melphalan concentrations. Liver function was assessed using standard blood tests and the indocyanine green-based test. No deaths related to the IHP procedure were recorded. All animals had transient liver function impairment, with all liver function test results returning to normal within the observation period. At histologic examination, liver damage was similar under both hyperthermic conditions. Melphalan levels in the perfusate were not significantly different in the two study groups (the mean perfusate/plasma area under the curve from 0 to 60 minutes ratios were 463 and 501, respectively). These results correlated well with those obtained using the scintigraphic method. Liver drug concentrations remained unchanged after true hyperthermia IHP. Under true hyperthermic conditions, neither an increase in liver parenchyma toxicity nor changes in melphalan pharmacokinetics were observed. These findings support the use of true hyperthermia in the clinical setting to exploit fully the antitumor synergism between melphalan and heat.     

Isolated hepatic perfusion for unresectable hepatic metastases from colorectal cancer

BACKGROUND: Unresectable colorectal liver metastases are a significant clinical problem. Isolated hepatic perfusion (IHP) is a regional treatment technique that delivers high dose chemotherapy, biologic agents, and hyperthermia via a completely isolated vascular recirculating perfusion circuit as a means of regionally treating liver tumors. This study presents our results of IHP with tumor necrosis factor (TNF) plus melphalan or IHP with melphalan alone followed by infusional floxuridine (FUDR) and leucovorin in patients with advanced or refractory unresectable hepatic colorectal metastases. METHODS: Fifty-one patients with unresectable colorectal hepatic metastases underwent a 60-minute IHP with 1.5 mg/kg melphalan and hyperthermia (39 degrees C to 40 degrees C). Thirty-two patients received IHP with 1 mg TNF with melphalan and 19 patients had IHP with melphalan alone followed by monthly hepatic intra-arterial infusional (HAI) FUDR (0.2 mg/kg/day) and leucovorin (15 mg/M(2)/day) for 14 days monthly for up to 12 months. Twenty-six patients failed 1 or more previous treatment regimens for established hepatic metastases and 27 had greater than 25% hepatic replacement (PHR) by tumor. Patients were monitored for response, toxicity, and survival. RESULTS: There was 1 perioperative death (2%), and only 2 patients (4%) had measurable perfusate leak during IHP (both less than 4%). In the 32 patients treated with IHP alone there were no detectable systemic TNF or melphalan levels during perfusion. The overall objective radiographic response rate (all partial [PR]) was 76% (38 of 50 assessable patients) with a median duration of 10.5 months (range, 2 to 21 months). Twenty-four of 31 patients (77%) had a PR after IHP alone and 14 of 19 (74%) after IHP with postperfusion HAI. Median duration of response was 8.5 months after IHP alone and 14.5 months after IHP and HAI; median survival was 16 and 27 months, respectively. There were 18 PRs in 26 patients (69%) whose prior therapy had failed and 18 PRs in 27 patients (67%) with PHR of 25 or greater. CONCLUSIONS: IHP can be performed with acceptably low morbidity and has significant antitumor activity in patients with unresectable hepatic metastases from colorectal cancer including those with refractory disease or PHR of 25 or greater. HAI appears to prolong the duration of response after IHP, and this combined treatment strategy deserves additional clinical evaluation as a therapeutic modality in this setting.     

初始灌注液对离体低温保存大鼠肝脏的影响

目的 探讨缺血再灌注过程中初始灌注液对离体低温保存大鼠肝脏的影响。方法采用离体大鼠肝脏低温缺血保存再灌注模型90例,分别选择UW液、HC-A液和乳酸林格液作为不同的初始灌注液,观察大鼠肝脏在低温保存0、3、6、12、24 h后再灌注流出液中ALT、AST、LDH和ET的水平,同时比较3组之间肝脏细胞形态和细胞凋亡。结果 使用HC-A液作为初始灌注液的大鼠肝脏再灌注流出液中ALT、AST、LDH和ET水平较其他两组低(P<0.05),肝脏形态和细胞凋亡的发生3组差异无显著性。另外,上述指标均受低温保存时间的影响。结论 初始灌注液可影响离体低温保存大鼠肝脏的质量,细胞凋亡可能参与了肝脏的缺血再灌注损伤。     

一氧化氮对全胃肠外营养肝脂肪变性的保护作用

目的探讨一氧化氮(NitricOxide,NO)对全胃肠外营养(TotalParenteralNutrition,TPN)引起的肝脂肪变性的作用。方法30只Wistar大鼠随机分为5组A组，正常对照；B组，TPN；C组，TPN+精氨酸；D组，TPN+N     

肝脏血管解剖与活体肝移植供者选择

目的 研究肝脏血管解剖对选择活体肝移植供者的影响.方法 总结分析312名健康成年人接受活体供肝移植供者评估的临床资料,其中男性278名,女性34名,年龄18～63岁,平均年龄29.3岁.所有受评估者均接受CT强化检查,并进行三维血管重建,观察肝脏血管走行,从而归纳出血管解剖对活体肝移植供者选择的影响.并观察其术后效果.结果 312名受评估者中,肝中动脉起源于肝左动脉的有218名(占69.9%).起源于肝右动脉的有89名(占28.5%),直接起源于肝总动脉的有5名(占1.6%).受评估者具有变异动脉的共有78名(占25%),其中具有副肝左动脉29名,具有副肝右动脉10名,具有替代肝左动脉29名,具有替代肝右动脉38人,其中多人同时具有两支变异动脉.门静脉的分型依据京都大学分型标准,门静脉I型有265名(占84.9%),Ⅱ型有25名(占8%),Ⅲ型有16名(占5.1%),Ⅳ型有6名(占2%),未发现门静脉V型者.Ⅳ段肝静脉汇入肝中静脉的有88名(占28.2%),汇入肝左静脉的为7名(占2.2%),汇入肝中静脉和肝左静脉的共有217名(占69.6%).共有221名受评估者具有右下静脉(占70.8%),其中89例具有两支及两支以上右下静脉.最终有194名受评估者作为肝移植供者接受了手术.结论 肝动脉具有较高的变异率;有Ⅰ～Ⅲ型门静脉者均可作为活体肝移植供者的选择条件;准确了解供肝血管解剖及正确的手术设计是保证活体肝移植供、受者手术成功的先决条件.     

Isolated hepatic perfusion for lapine liver metastases: impact of hyperthermia on permeability of tumor neovasculature

BACKGROUND: Hyperthermic isolated hepatic perfusion (IHP) has been shown to cause significant regression of advanced unresectable liver metastases in patients. Although there are different agents and treatment modalities used in IHP, the contribution of perfusion hyperthermia is unknown. PURPOSE: A large animal model of unresectable liver metastases and a technical standard for IHP in this model were established. This model was used to assess the effects of hyperthermia on vascular permeability of tumors and normal liver tissue during IHP. METHODS: Sixty-five New Zealand White rabbits were used in a series of experiments. Disseminated liver tumors were established by direct injection of 1 x 10(6) VX-2 cells into the portal vein by laparotomy in anesthetized animals. Several surgical perfusion techniques were explored to determine a reliable and reproducible IHP model. Vascular permeability in tumor versus liver was then assessed with Evan's Blue labeled bovine albumin under normothermic (tissue temperature 36.5 degrees C +/- 0.5 degree C), moderate hyperthermic (39 degrees C +/- 0.5 degree C), or severe hyperthermic (41 degrees C +/- 0.5 degree C) conditions. RESULTS: Tumor model and perfusion techniques were successfully established with inflow through the portal vein and outflow through an isolated segment of the inferior vena cava. A gravity driven perfusion circuit with stable perfusion parameters and complete vascular isolation was used. Vascular permeability was higher in tumor than in normal tissues (P = .03) at all time points during IHP. Hyperthermia resulted in a significant (up to 5-fold) increase in permeability of neovasculature; when severe hyperthermia was used, tumor vascular permeability was increased even more than normal liver permeability (P = .01). CONCLUSIONS: The VX-2/New Zealand White rabbit system can be used as a reproducible large-animal model for IHP of unresectable liver metastases. It can be used to characterize the contribution and mechanism of action of different treatment parameters used in IHP. Hyperthermia preferentially increases vascular permeability in tumors compared with liver tissue in a dose-dependent fashion, thus providing a mechanism for its presumed benefit during isolated organ perfusion.     

大鼠肝移植术后肝脏T淋巴细胞凋亡与免疫耐受的关系

目的探讨肝移植术后肝组织T淋巴细胞凋亡和肝移植免疫耐受之间的关系。方法采用Kamada二袖套法建立Wistar→Sprague-Dawley(SD)原位肝移植(OLT)大鼠模型。实验动物随机分为3组,每组各6只。A组:空白对照组,不作任何处理,采用SD大鼠;B组:免疫排斥组,Wistar大鼠为供体,SD大鼠为受体,行OLT;C组:免疫耐受组,Wistar大鼠为供体、SD大鼠为受体,行OLT,术前1周胸腺内注射F蛋白0.4mg,建立稳定的移植耐受大鼠模型。A组立即处死大鼠,B组和C组分别于术后7d、100d处死大鼠取肝组织,分别取各组大鼠肝组织标本进行冰冻切片,应用原位末端脱氧核苷酸转移酶标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)在荧光显微镜下检测肝移植术后肝脏T淋巴细胞的凋亡情况。各组样本另取一张切片行常规苏木素-伊红(HE)染色在光学显微镜下观察,与TUNEL荧光染色法作对比观察。结果光学显微镜下,B组可见中、重度免疫排斥反应表现,C组肝组织细胞间的淋巴细胞浸润较B组大大减少,稍多于A组。荧光显微镜下,A组TUNEL切片可见零星散在的凋亡细胞,C组可见大量散在或密集分布的凋亡细胞,B组的凋亡细胞数远较C组减少,但仍多于A组。A组、B组、C组大鼠肝组织内浸润T淋巴细胞的凋亡指数(apoptosisindex,AI)分别为(8.83±0.43)%、(11.32±1.29)%和(19.00±1.96)%,两两比较差异有统计学意义(P<0.05～0.01)。结论免疫耐受移植物内浸润的T淋巴细胞凋亡明显增高,浸润的T淋巴细胞凋亡受阻可能会阻碍免疫耐受的发生,引起排斥反应。     

Isolated hyperthermic liver perfusion with high dose tumor necrosis factor alpha in pigs: an experimental study in preparation of clinical Use

Isolated hyperthermic perfusion of the liver was performed for 45 min in 27 pigs via hepatic artery and portal vein at mean inflow temperatures between 40.7 and 41.2 degrees C. In two study groups B and C (n = 9 pigs each) 50 microg recombinant human tumor necrosis factor-alpha (rhTNFalpha) per kg body weight were added to the perfusate, whereas in a control group A liver perfusion was done without rhTNFalpha. Before reperfusion the livers were washed out with Ringer's solution in all groups followed by a protein solution in group C. At 30 and 60 min after reperfusion the maximum systemic rhTNFalpha concentrations were significantly higher in group B with 68 and 61 ng/ml compared to 14.5 and 14.9 ng/ml in group C (p < 0. 05). Mean systemic porcine TNFalpha concentration was significantly higher in group B (217 pg/ml) compared to group C (50 pg/ml) 30 min after reperfusion (p = 0.012). Survival was 7/9 in group A and C and only 2/9 in group B with 6/7 pigs dying due to severe cardiopulmonary failure within 12 h after operation. In surviving pigs of group A and C only mild and transient hepatotoxicity was registered. The presented study underlines the feasibility of high dose rhTNFalpha application in an isolated hyperthermic liver perfusion system. Washout of the liver with a protein solution before reperfusion reduces systemic TNFalpha levels as well as associated lethal cardiocirculatory and hepatotoxic side effects.