【作者】 柴军武；

【导师】 沈中阳； 郑虹； 蒋文涛；

【作者基本信息】 天津医科大学 ， 外科学， 2009， 硕士

【摘要】 【目的】供肝短缺严重地制约着临床肝移植的发展。随着活体肝移植技术的不断成熟,其已成为缓解供体短缺的重要途径之一。临床活体肝移植术后早期常应用肝素、低分子肝素钠或低分子肝素钙来预防或治疗肝动脉血栓形成,而肝素对活体肝移植术后移植肝脏和供体残余肝脏再生的影响缺乏相关研究。本实验在大鼠减体积肝移植模型的基础上,初步探讨肝素对肝再生的影响。【方法】实验前期进行显微外科训练,尝试二袖套法建立大鼠全肝肝移植模型和系列减体积肝移植(60%、50%、30%)模型。在此基础上选取同季节健康雄性wistar大鼠120只(北京军事医学科学院实验动物中心),体重230-280g,随机分为四组(随机数表法):A、B两组每组42只,供受体随机选取,C、D两组每组18只。A、B两组行50%减体积肝移植术后分别给予生理盐水(4ml/kg)、肝素(1000IU/kg),C组行50%肝切除术后给予生理盐水(4ml/kg),D组为假手术组,术后给予生理盐水(剂量同C组),各组均为术后20h皮下给药。各组大鼠术后3天、5天、7天随机牺牲6只。牺牲鼠麻醉开腹后下腔静脉取血5ml,静置后离心取血清,酶联免疫吸附试验检测肝细胞生长因子(HGF);取移植肝脏,记录牺牲前大鼠重量、肝脏湿重,计算肝再生速率,取部分肝脏制作肝脏单细胞悬液检测肝细胞增殖指数,部分肝脏-80℃深低温保存,剩余肝脏甲醛固定,做常规HE染色和免疫组化染色(肝细胞增殖核抗原PCNA),记录PCNA表达情况。统计学分析比较A与B组、A与C组肝脏再生指标的差异(SPSS13.0,独立样本t检验),比较A与B组、A与C组HGF水平的差异。【结果】前期完成一百余例大鼠全肝肝移植和系列减体积肝移植,建立了稳定的大鼠肝移植模型。A、B两组大鼠50%减体积肝移植术中供体切取时间分别为30±3min、31±4min,无肝期分别为15±2min、16±2min,冷缺血时间分别为56±5min、54±7min,受体手术时间分别为54±7min、58±5min。A、B两组术后48h存活率分别为95.2%(20/21)、100%(21/21),移植鼠无出血和感染死亡,A组两例因术后发生胆道并发症排除,胆道并发症发生率为10%(2/20),B组3例因术后发生胆道并发症排除,胆道并发症发生率14.3%(3/21)。C组完成大鼠50%肝切除18例,手术用时(14±2)min。C、D两组无死亡和并发症病例。A、B、C三组术后3天、5天、7天肝再生速率分别为(33.5±6.6)%、(43.7±5.4)%、(45.6±6.3)%,(40.1±2.8)%、(71.9±17.8)%、(56.9±5.4)%,(86.6±11.9)%、(111±19.2)%、(98.4±4.6)%。A、B两组术后3天、5天、7天肝再生速率比较均有统计学差异(P=0.015＜0.05、P=0.002＜0.01、P=0.023＜0.05)。A、C两组术后3天、5天、7天肝再生速率比较均有统计学差异(P=0.012＜0.05、P=0.011＜0.05、P=0.031＜0.05)。A、B、C组大鼠术后3天、5天、7天血清HGF水平分别为(263.3±101.1)pg/ml、(420.7±56.8)pg/ml、(365.3±6.4)pg/ml,(312.9±10.1)pg/ml、(440.9±23.4)pg/ml、(384.4±32.2)pg/ml,(242.4±22.1)pg/ml、(409.9±23.0)pg/ml、(376.3±22.3)pg/ml。A、B两组大鼠术后3天、5天、7天血清HGF水平比较有统计学差异(P=0.008＜0.01、P=0.000＜0.01、P=0.000＜0.01)。A、C两组术后3、5、7天血清HGF水平比较均有统计学差异(P=0.016＜0.05、P=0.038＜0.05、P=0.001＜0.01)。A、B、C组术后3天、5天、7天肝细胞增殖指数PI分别为:(30.4±4)%、(48.1±8)%、(38.4±2)%,(29.3±3)%、(43.5±8)%、(36.9±2)%,(30.3±3)%、(47.3±8)%、(31.6±3)%。A、B两组不同时间段PI指数水平比较均有统计学差异(P=0.001＜0.01、P=0.002＜0.01、P=0.02＜0.05)。A、C两组术后3、5天PI指数比较均有统计学差异(P=0.025＜0.05、P=0.018＜0.05),A、C两组术后7天PI指数比较无统计学差异(P=0.86＞0.05)。A、B两组术后3天、5天PCNA表达均有统计学差异,而术后7天PCNA表达无统计学差异。A、C两组术后3天、7天PCNA表达无统计学差异,术后5天PCNA表达有统计学差异。【结论】皮下给予肝素(1000IU/kg)可促进大鼠50%减体积肝移植术后肝再生;大鼠减体积肝移植术后肝脏再生速率较肝切除术后肝再生延缓;本实验肝素促进血清HGF水平的升高可能是肝素促进肝再生的机制之一。更多还原

【Abstract】 【Objective】Unfractionated heparin,low molecular weight heparin were regularly utilized to prevent or treat hepatic artery thrombosis during early postoperative in clinical living donor liver transplantation.However,there is few literatures on effects of heparin on hepatic regeneration after 50%partial liver transplantation in rats.This study was designed to investigate effects of unfractionated heparin on liver regeneration in rats after reduced-size liver transplantation.【Methods】Try to set up stable Orthotopic Liver Transplantation model and serial reduced-size liver transplantation(60%,50%,30%) model in rats using two-cuff technique.To master the surgical skills of Liver transplantation in rats and reduce the complications.On this basis to select the same season,the same batch healthy male wistar rats 120(Beijing Academy of Military Medical Sciences),weighting 230-280g, were randomly divided into four groups(random number table method):A,B two sets were 42 rats each group,C,D were 18 in each group,the donors and receptors were randomly selected.50%reduced-size orthotopic liver transplantation was performed in A,B groups using two-cuff technique.The rats in A,B groups were given normal saline(4ml/kg) and unfractionated heparin(1000U/kg) respectively. 50%hepatectomy was performed in C group given saline after hepatectomy(4ml/kg). D group was sham-operated group,given normal saline(dose with the C group) postoperative.All of the drugs were administrated subcutaneous 20h postoperative. Rats in each group after 3 days,5 days,7 days were randomly sacrificed 6.Blood samples(5ml) were obtained from inferior vena cava of rat when Rats were sacrificed after anesthesia.Get serum after standing and centrifugalization,then put serum in the refrigerator(-80℃),and detect hepatocyte growth factor(HGF) by enzyme-linked immunosorbent assay(ELISA);check transplanted liver,Record rat weight,liver wet weight,the calculation of liver regeneration rate,make single-cell suspension from part of a liver by mechanical method;Detect Liver cell proliferation index by flow cytometer,part of a liver cryopreserved -80℃,and the remaining part of liver was fixed by formaldehyde,make conventional HE staining and immunohistochemical staining(liver cells proliferating nuclear antigen PCNA),Record PCNA expression. Results were analyzed using SPSS 13.0 statistical analysis software,observe compa rison of various indicators of group differences in liver regeneration(independent samples t test),and compare each group differences in the levels of HGF.【Results】Complete one hundred cases of orthotopic liver transplantation in rat and a series of reduced-size liver transplantation(60%,50%,30%) before formal experiment,and set up a stable model of rat liver transplantation.Rats of group A and B were performed 50%reduced-size liver transplantation,the time for donor operation was(30±3)min and(31±4)min respectively,the recipient operation time was(54±7)min and(58±5)min,respectively,the anhepatic time was(15±2)min and(16±2)min,cold ischemia time was(56±5)min、(54±7)min respectively,the 48 hours survival rate was 95.2%(20/21)and 100%(21/21),no rats died of infection and bleeding.Two rats of group A were excluded due to biliary complications of postoperative,the incidence of biliary complications was 10%(2/20).Three cases of group B were excluded due to biliary complications of postoperative,the incidence of biliary complications was 14.3%(3/21).The rats of group C(18 cases ) were completed 50%hepatectomy,time for the operation was(14±2)min.There was no case of death and complications of group C and D.Liver regeneration rate of groups A,B,C on the third,fifth,seventh day after surgery was(33.5±6.6)%、(43.7±5.4)%、(45.6±6.3)%,(40.1±2.8)%、(71.9±17.8)%、(56.9±5.4)%,(86.6±11.9) %、(111±19.2)%、(98.4±4.6)%,respectively.There were significant differences(P = 0.015 <0.05,P= 0.002 <0.01,P= 0.023 <0.05) between A and B on the third,fifth, seventh day after surgery.There were significant differences(P=0.012<0.05,P= 0.011<0.05,P=0.031<0.05) between A and C on the third,fifth,seventh day after surgery.Serum HGF levels of groups A,B,C on the third,fifth,seventh day after surgery were(263.3±101.1)pg/ml、(420.7±56.8) pg/ml、(365.3±6.4)pg/ml, (312.9±10.1)pg/ml、(440.9±23.4)pg/ml、(384.4±32.2)pg/ml,(242.4±22.1) pg/ml、(409.9±23.0)pg/ml、(376.3±22.3)pg/ml.There were significant differences on serum HGF levels on the third,fifth,seventh day after surgery(P = 0.008 <0.01,P = 0.000 <0.01,P = 0.000 <0.01) between A and B.There were significant differences on serum HGF levels on the third,fifth,seventh day after surgery(P=0.016<0.05,P=0.038<0.05,P=0.001 <0.01) between A and C. Hepatocyte PI index of groups A,B,C on the third,fifth,seventh day after surgery were as follows:(30.4±4)%、(48.1±8)%、(38.4±2)%、(29.3±3)%、(43.5±8) %、(36.9±2)%,(30.3±3)%、(47.3±8)%、(31.6±3)%,there were significant differences on hepatocyte PI index on the third,fifth,seventh day after surgery(P = 0.001 <0.01,P = 0.002 <0.01,P = 0.02 <0.05)between A and B,and significant differences(P=0.025<0.05、P=0.018<0.05) on the third,fifth day between A and C,but have no difference on the seventh day between A and C(P=0.86>0.05).There were differences on PCNA expression between A and B on the third,fifth day,but no significant difference on PCNA expression on the seventh day.There were differences on PCNA expression between A and C on the fifth day,but no significant difference on PCNA expression on the third,seventh day.【Conclusion】unfractionated heparin(1000IU/kg) given subcutaneous significantly promote hepatic regeneration after 50%reduced-size liver transplantation in rats;the liver regeneration of 50%reduced-size liver transplantation was delayed comparing to hepatectomy.In this experiment,it may be one of the mechanisms beneficial to liver regeneration that heparin elevates the plasma production of hepatocyte growth factor.更多还原