MnTBAP对小体积肝移植物缺血再灌注损伤保护作用的研究

【作者】 崔一尧；

【导师】 钱建民； 芮晓晖；

【作者基本信息】 复旦大学 ， 外科学， 2009， 博士

【摘要】 第一部分不同方法制作小体积大鼠肝移植模型的比较目的：在对大鼠肝脏移植模型熟练掌握的基础上，根据近年来在“三袖套法”与“二袖套法”在制作模型方面的进展，比较“三袖套法”与“二袖套法”在单人制作模型方面的优缺点，为进一步完成实验研究提供手术成功率高的大鼠模型。方法：利用一些自制简单工具，建立大鼠30％非动脉化大鼠原位肝移植模型。实验分为2组：Ⅰ组：以Miyata M等人的“三袖套法”制作大鼠中叶供肝的30％中叶供肝小体积肝移植组(n=15)；Ⅱ组：参照Kamada等人的“二袖套法”制作大鼠30％中叶供肝小肝移植组(n=15)。对Ⅰ、Ⅱ组手术操作时间、受体无肝期、手术成功率、1周存活率及并发症发生率进行分析。结果：Ⅰ组肝上下腔静脉套管失败2例，手术成功率86．6％；Ⅱ组手术成功率100％。Ⅰ、Ⅱ组切取供肝平均时间分别为：Ⅰ组，67．75±7．124min；Ⅱ组，46．00±5．940min；2组间有统计学差异(P＜0．05)；受体切除肝脏时间：Ⅰ组，36．27±11．074min；Ⅱ组，16．13±1．922min,2组间存在统计学差异(P＜0．05)。无肝期时间：Ⅰ组，9．92±2．021min；Ⅱ组15．87±2．588min；2组间存在统计学差异(P＜0．05)。Ⅰ组和Ⅱ组大鼠术后1周存活率均达到40％，组间无统计学差别。并发症发生率低，2组间无统计学差异，模型稳定。结论：我们对套管的工具进行了一定的改进，在辅助工具下行“三袖套法”建立大鼠肝移植模型，其肝上下腔静脉套管困难的不足虽然得到一定程度的克服，但仍然有一定的失败率，而且供体修剪时间长，冷缺血时间明显延长。相对而言，“二袖套法”的大鼠肝移植模型冷缺血时间短；无肝期时间虽然比较长，但由于技术的进步，都控制在25分钟内，生存率不低于“三袖套法”建立的大鼠肝移植模型。“二袖套法”技术要求稍低，更容易掌握，训练时间不是太长，手术成功率高。熟练掌握大鼠肝移植技术，改进套管方法，并注重所有手术细节的处理，单人采用“二袖套法”建立稳定可靠的大鼠肝移植模型，更具可行性。第二部分MnTBAP对不同体积肝移植大鼠术后肝细胞存活的影响目的：探讨MnTBAP对SD大鼠不同体积肝移植术后肝细胞存活的影响及其机制。方法：参照Kamada等人的“二袖套法”建立不同体积SD大鼠原位肝移植模型，实验分为四组，60％体积肝移植组(A组)、45％体积肝移植组(B组)、30％体积肝移植组(C组)和假手术组(D组)。前3组又分为单纯移植组和MnTBAP治疗组。比较拟于术后3h处死的各组大鼠，在断流前及门静脉再灌注后即刻、15分钟、30分钟门静脉压力变化；收集受体再灌注后3h、6h、12h和24h、48h外周血查ALT变化、肝脏中核转录因子-κB(NF-κB)及其抑制物I-κB、肿瘤坏死因子—a(TNF-a)的表达、丙二醛(malondialdehvde，MDA)、与肝细胞损伤间的关系。病理学检查肝细胞坏死及凋亡的变化情况、并观察术后7天生存率。结果：大鼠肝移植术后门脉复流早期，门静脉的压力在30％小肝组较45％及60％肝移植组明显升高(即刻、15min，P＜0．05)；并且45％小肝组门静脉压力也高于60％肝移植组，但统计学并无差异(P＞0．05)。各移植组肝组织NF-κB的活性在复流后3、6、12h明显升高(P＜0．05)；TNF-a及MDA含量在6、12、24h显著升高(P＜0．05)，血清丙氨酸转氨酶(ALT)活性亦显著升高(P＜0．05)，ALT水平在小肝组明显高于60％肝移植组(P＜0．05)，两个小肝组之间无差别(P＞0．05)。病理显示组织损伤、肝血窦扩张在小移植物组较60％肝移植组细胞损伤变化更重(P＜0．05)；I-κB在3、6、12h的表达均下降(P＜0．05)。与同组中单纯移植组比较，各组中经MnTBAP治疗组的门静脉压力较单纯移植组无明显变化(P＞0．05)，但各组中经MnTBAP治疗组的血清ALT水平(P＜0．01)、NF-κB的活性(P＜0．05)、肝组织TNF-a的表达均有显著下降(P＜0．05)，且MDA含量明显降低(P＜0．05)。病理检查显示：肝细胞损伤变化减轻。结论：肝移植术后，门静脉的压力与移植物体积大小密切相关。ROS的生成与门静脉的压力、肝损伤的程度密切相关。MnTBAP不能降低门静脉压力，但是对ROS的生成有明显抑制作用，减轻了ROS促进损伤性细胞因子表达的作用，减轻了移植肝的I/R损伤。第三部分MnTBAP对大鼠不同体积肝移植术后肝再生的影响目的：研究MnTBAP对不同体积大鼠小肝移植物模型肝再生的作用及机制。方法：参照Kamada等人的“二袖套法”建立不同体积SD大鼠原位肝移植模型，实验分为6组，60％体积肝移植组，A组(n=30)；45％体积肝移植组，B组(n=30)；30％体积肝移植组，C组(n=30)；经MnTBAP治疗的60％体积肝移植组，D组(n=30)；经MnTBAP治疗的45％体积肝移植组，E组(n=30)；经MnTBAP治疗的30％体积肝移植组，F组(n=30)。比较受体再灌注后3h、6h、12h和24h、48h外周血查ALT变化、肿瘤坏死因子—a(TNF-a)、IL-6水平的表达；检测不同时间段移植物损伤、肝细胞增殖、相关的细胞周期蛋白的表达、TNF-a/IL-6/Stat3通路的激活情况及相互关系。病理学检查肝细胞再生的变化情况、并观察术后7天生存率。结果：SD大鼠肝移植后移植物内TNF-a、IL-6水平迅速升高，分别于术后6～12h达高峰。与A组比较，B、C组移植物内TNF-a、IL-6水平显著升高(P＜0．01)，且C组高于B组，但B、C组间并无统计学差异(P＞0．05)；复流后6、12、24h，移植物内TNF-a、IL-6水平在各MnTBAP治疗组的表达与同体积单纯肝移植组比较，均存在统计学差异(P＜0．05)，但TNF-a、IL-6水平在各MnTBAP治疗组的表达仍维持较高水平。与同体积单纯肝移植组比较，Stat3/PStat3、细胞周期蛋白cyclinD1表达、PCNA阳性细胞比率在D、E组的结果均无统计学差异(P＞0．05)。但与同体积30％单纯肝移植组比较，Stat3/PStat3、细胞周期蛋白cyclinD1表达、PCNA阳性细胞比率在F组的结果却存在统计学差异(P＜0．05)。总体上，MnTBAP治疗组与单纯肝移植组比较，其术后7天生存率存在统计学差别(P＜0．05)。结论：由于多种因素影响，30％肝移植组肝脏再生能力在复流后早期受损：MnTBAP治疗可以降低小移植物肝组织损伤程度，对肝细胞再生有良好的保护作用，可能与维持TNF-a介导的stat3/Pstat3通路的稳定有关。更多还原

【Abstract】 PartⅠDifferent methods to establish rat small-for-size liver transplantation model bycomparisonObjective:to explore an effective and viable way to establish small-for-size livertransplantation model in the rat by single person on the basis of skilled grasp of thesmall-for-size liver transplantation model.Methods:Male SD rats are randomly divided into the following groups as donors andrecipients:Group A:According to Miyata M,15 rats for to establish 30% size graft livertransplantation using a median lobe graft by“three-cuff”techniques;Group B(n=15):30% sizegraft liver transplantation rat model using a median lobe graft by Kamada’s“two-cuff method”on the basis of skilled grasp of the small-for-size liver transplantation model;Time for surgicalprocedure including duration of donor-graft preparation,recipient operation and anhepaticphase,7-day survival rate and the technical complications are compared between the twogroups.Results:Compared to Group B,the times for donor-graft preparation,recipient operationin Group A is longer (P＜0.05) ,the mean times to perform SHVC anastomoses and anhepaticphase is less (P＜0.05) but higher in failure rate to complishment of the connection (P＞0.05).Compared Group B,Surgical complications in group A show no significant difference(P＞0.05) .There are no significant differences in the 7-day survival rate between twotransplantation groups (P＞0.05) ,though Group B has more survivors 7 days after livertransplantation.Conclusion:With improved method of cuff connection,more effective and viable 30%small-for-size rat liver transplantation model with two-cuff method can be established stablly. PartⅡProtective effects of MnTBAP on small graft for size of liver transplantation afterischemia reperfusion injury in ratsObjective:To observe the protective effects of MnTBAP on small graft for size of livertransplantation after ischemia reperfusion (I/R) injury in rats and to find out its possiblemechanism.Methods:Male SD rats are randomly divided into three groups,which were shamoperation group,untreated group and MnTBAP treated group,and both untreated group andMnTBAP treated group contain different size graft animals including 60%LDLT,45%LDLTand 30%LDLT.Alanine aminotransferase (ALT) in rats after 3h,6h,12h,24h and 48h afterreperfusion are evaluated,and the activity of superoxide dismutase (SOD) and the content ofmalondialdehyde (MDA) in ischemia small graft for size are examined.The apoptotic index(AI) of hepatocytes in small graft for size is detected by terminal deoxyribonucleo -tidyltransferase (TdT)mediated dUTPbiotin nick end labeling (TUNEL) methods.The expressionof TNF-a and NF-κB are examined by ELISA methods.Results:Even though the pressure of portal vein has no difference between untreatedgroup and MnTBAP treated group(P＞0.05),But serum ALT in rats after 3h,6h,12h,24h,48hafter reperfusion in MnTBAP treated group are obviously lower as compared with those in theuntreated group (P＜0.01),and MDA in MnTBAP treated group are lower VS.those in theuntreated group (P＜0.05);The decreased apoptotic index and the expression ofdownregulated TNF-a and NF-κB are also observed in MnTBAP treated group in 6h,12h,24hand 48h after reperfusion(P＜0.05).In untreated group,the expression of TNF-a is highestamong the three groups(P＜0.05),and the serum ALT(P＜0.01) and MDA level is highesttoo(P＜0.05).The hepatocellular apoptosis is more serious than that in other groups(P＜0.05).Conclusion:The protective effects of MnTBAP against I/R injury of small graft for sizemay associate with highly scavenging reactive oxygen species(ROS),reducing lipidperoxidation injury and inhibiting apoptosis.which may due to the smaller graft size,refusionwith higher portal pressure,and subsequent hepatic parenchymal injury. PartⅢEffect on Liver Regeneration of Small for Size Graft in Liver Transplantation RatsAdministered MnTBAPObjective:To study the protective effect and the possible mechanism on liverregeneration of small for size graft in liver transplantation rat models administered MnTBAPvia peritoneal cavity.Methods:Male Sprague-Dawley rats are divided into six groups for three groups(n = 30in each group)of liver transplantation models with graft volume / standard liver volumerespectively 60%,45 % and 30% which are untreated group by MnTBAP ;Other three groups(n= 30 in each group)of liver transplantation models with the same graft volume / standard livervolume which are treated by MnTBAP group.The expression of TNF-α,IL-6,Stat3/Pstat3,JNK and the level of malondialdehyde(MDA) are detected as well as alanineaminotransferase(ALT) level at 3,6,12,24h and 48h after reperfusion.Proliferating CellNuclear Antigen (PCNA) is detected by immunohistochemical methods.The morphologicalchanges of hepatic tissue were observed through light microscope at different time points.Result:Compared with the same volume graft of untreated group,That the graftexpress TNF-αand the level of IL-6 are significantly lower (P＜0.05) in all treated groups byMnTBAP and these animals exhibit improved liver function and liver weight recovery duringthe early post-transplantation.In liver transplantation model groups treated by MnTBAPexcept 30% with graft volume / standard liver volume,the graft express JNK and Pstat3 andare no significantly differance in 6,12,24h after refusion than that in untreatedgroup(P＞0.05),meanwhile compared with the same volume graft of untreated group,thepercentage of PCNA is almost at the same level in all groups except that in 30% livertransplantation untreated group(P＜0.05).Conclusion:MnTBAP can decrease the activities of ROS and the release ofinflammatory mediators to protect the graft against ischemia / reperfusion injury but canfurther mantain JNK activation by highly durative expression of IL-6 and TNF-α.It may offera novel approach to protect liver regeneration after small-for-size-graft transplantation.更多还原