【作者】 程峰；

【导师】 程惊秋；

【作者基本信息】 四川大学 ， 遗传学， 2004， 博士

【摘要】 目的：肝脏缺血再灌注损伤是肝移植不可避免的一个过程，也是一种重要的非免疫学因素，严重时可引起移植肝脏失功、其它器官损伤甚至病人死亡。因此，缺血再灌注损伤严重影响移植肝脏的存活质量，寻找有效的干预措施是肝移植面临的一项重要课题。 缺血再灌注损伤还可通过上调移植相关抗原的表达，加剧移植排斥反应，加速移植物的慢性失功。大量证据表明MHC Ⅰ类分子相关抗原(MIC)与移植物排斥密切相关，但是缺血再灌注损伤能否引起MIC的表达尚无报道。 本文在前期研究基础上进一步研究益生注射液(Yisheng Injection，YS)抗肝脏缺血再灌注损伤的作用机制以及肝脏缺血再灌注损伤对小鼠MIC(RAE-1、H60)基因表达的影响和益生注射液的干预效果。 方法：采用小鼠部分肝脏热缺血再灌注损伤模型研究益生注射液的干四川大学博士学位论文预机理以及RAE一1、H60基因的表达。 l)C57BL/6小鼠随机分为5组:假手术组(Sham)、模型组(I瓜)、YS小剂量(5m眺g)干预组、YS中剂量(10m酬kg)干预组和YS大剂量(ZOmg/kg)干预组。缺血前24小时和1小时YS干预组小鼠腹腔注射分别给予5、10或20mg/kg的YS，钳夹肝左叶造成部分肝脏缺血gomin，再灌注时经尾静脉注射第三次给予同样剂量的YS，模型组和假手术组给予同样体积的生理盐水。 小鼠肝脏左叶缺血90min再灌注6h后各实验组采集血液和肝脏左叶组织样本。检测血清中丙氨酸氨基转移酶(ALT)、门冬氨酸氨转移酶(AST)和乳酸脱氢酶(LDH)的活性作为肝脏损伤的指标，同时用ELISA检测血清中TNF一。的含量。检测肝组织中髓过氧化物酶(MPo)、超氧化物歧化酶(SOD)和丙二醛(MDA)含量以及肝脏水肿程度。肝脏组织病理学检测。RT-PCR和免疫组化检测肝组织中INF一Q和ICAM一1的表达。 2) BALB/C小鼠随机分为2组:假手术组(S ham)和模型组(I瓜)。模型组小鼠肝脏左叶缺血90min再灌注。假手术组(s ham)和模型组(I瓜)手术后l、2、3、5、7、10、15、20、30天肝脏左叶组织分别取样。采用Rr-pCR和real一time pCR检测RAE一l、H6o基因在各实验组的表达。 3)BALB/C小鼠随机分为3组:假手术组(sham)、模型组(I瓜)和YS大剂量(20mg/kg)干预组。模型组和YS干预组小鼠肝脏左叶缺血90min再灌注。缺血前24小时和1小时YS干预组小鼠腹腔注射给予20mg瓜g的YS，再灌注时经尾静脉注射第三次给予同样剂量的YS。再灌注后YS干预组每天给予20m留kg的YS直至再灌注后第7天。模型组和假手术组给予同样体积的生理盐水。手术后7天肝脏左叶组织分别取样，采用real·time PCR检测RAE一1、H60基因在各实验组的表达。结果:l)益生注射液干预能有效降低小鼠部分肝脏热缺血90min再灌注6h所致的血清ALT、AST、LDH水平升高，降低血清中飞NF一a含量，四川大学博士学位论文干预后血管内皮、肝窦内皮和肝细胞形态结构相对正常，血管和胆管周围淋巴细胞浸润减轻。 2)益生注射液干预能有效减轻缺血再灌注所致肝脏过氧化损伤，提高缺血肝脏中SOD酶活性、降低MDA含量。 3)益生注射液干预能有效降低缺血肝脏中MPO含量，减轻中性粒细胞的聚集和浸润，减轻缺血肝脏的水肿程度。 4)益生注射液干预能有效抑制缺血肝脏中TNF一a和ICAM一1分子在mRNA水平和蛋白质水平的高表达。 5)益生注射液(5、10、20mg瓜g)干预肝脏热缺血再灌注损伤具有良好的剂量一效应关系。 6) BALB/C小鼠正常肝脏组织能检测到RAE一1低表达。小鼠部分肝脏热缺血90min再灌注，可造成缺血肝脏中RAE一1 mRNA水平从再灌注第5天开始显著升高，巧天达峰值，一直持续到再灌注后30天，比对照组升高3一8倍。 7)BALB/C小鼠正常肝脏组织未检测到H60的表达。肝脏热缺血90min再灌注，可造成缺血肝脏中H60 mRNA水平从再灌注第1天开始显著升高，第5天达峰值，以后逐渐下降，直到再灌注后10天，比对照组升高2.5一8.5倍。但从再灌注15天开始到再灌注后30天，缺血肝脏的H6O mRNA水平与对照组相比无显著性差异。 8)益生注射液(2 omg/kg)干预可显著降低缺血肝脏中RAE一1、H60mRNA水平。干预组缺血肝脏中RAE一1、H60 mRNA水平与I瓜组相比分别下降70%(P<0.01)和76%(P<0.01)。结论:益生注射液干预能减少肝脏缺血再灌注后氧自由基的产生，减轻脂质过氧化引发的肝组织损伤;降低中性粒细胞浸润和肝脏水肿程度;有效抑制缺血再灌注损伤引起的TNF一a、ICAM一1 mRNA水平上调，降低下NF一a和ICAM一1在肝组织中的高表达;具有良好的剂量一效应关系。四川大学博士学位论文 小鼠肝脏缺血再灌注损伤可上调缺血肝脏中RAE一1、H60 mRNA水平，且二者上升时相不同。益生注射液干预可显著降低缺血肝脏中RAE一l、H60 mRNA水平。关键词:肝脏缺血再灌注损伤，益生注射液，RAE一1基因，H60基因， 荧光定量PCR更多还原

【Abstract】 Objective: Hepatic ischemia/reperfusion injury (IRI) is an important nonimmunologic factor and remains a significant problem and limitation of liver transplantation and may result in liver failure, remote organ failure, and even death. For this reason, the protection of liver against ischemia/reperfusion injury remains one of the major nonimmunologic problems of liver transplantation.In addition, hepatic ischemia/reperfusion injury may increase the expression of some antigens associated with allograft rejection, such as MHC class I and MHC class II molecules, and thus make the allografts more " immunogenic" and therefore more susceptible to rejection and dysfunction. Increaseing evidences demonstrate that MHC class I chain-related antigen A (MICA) and B (MICB) may play a role in allograft rejection, however, whether IRI affects the expression of MIC has not been documented.Our previous studies demonstrate that Yisheng injection (YS), a herbal preparation developed from traditional Chinese medicine, can attenuate ischemia/reperfusion injury in vitro and in vivo. Based on the previous results, the present study explored the effect and mechanism of YS on hepatic ischemia/reperfusion injury; determined the effect of IRI on the expression of RAE-1 (retinoic acid early inducible) and H60 genes, which are the functional homologs of human MIC in mouse; and examinated the effect of YS intervention on the expression of RAE-1 and H60 genes.Methods: Mice partial hepatic warm ischemia/reperfusion model was used in this study.1. C57BL/6 mice were randomly assigned into five experimental groups as follows: (1) sham operation group, (2) I/R group, (3) small-dose (5mg/kg of YS) treated group, (4) middle-dose (10mg/kg of YS) treated group, (5) large-dose (20mg/kg of YS) treated group.YS with different doses (5, 10, 20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. The sham and I/R group received same volume of sterile saline. After 90 min of ischemia and 6 h of reperfusion mice were sacrificed and liver tissue (from the ischemic lob) and blood samples were taken for analysis. The ALT, AST and LDH levels in the sera were measured, and the levels of SOD, MDA, MPO and edema of ischemic liver were examinated. The serum level of TNF- a was assayed by enzyme-linked immunosorbent assay (ELISA), and the expression of TNF-a and ICAM-1 was determined by immunohistochemical assay and reverse transcription polymerase chain reaction (RT-PCR).2. BALB/C mice were randomly assigned into sham and I/R groups. After90 min of ischemia the left lob of the liver in the I/R group was reperfused. Mice were sacrificed at intervals of 1, 2, 3, 5, 7, 10, 15, 20, and 30 days postoperatively, and the normal, sham and ischemic liver tissues were collected. The expression of RAE-1 and H60 genes was measured by RT-PCR and real-time quantitative PCR.3. BALB/C mice were randomly assigned into three groups as follows: (1) sham operation group, (2) I/R group, (3) YS (20mg/kg) treated group. After 90 min of ischemia the left lob of the liver in the I/R and YS treated groups was reperfused. YS (20 mg/kg) was injected intraperitoneally 24 h and 1 h before ischemia and a third dose was injected intravenously just before reperfusion. After operation YS treated group was given YS (20mg/kg/d) for 7 days. The sham and I/R group received same volume of sterile saline. Mice were sacrificed at 7 days after operation, and the normal, sham, ischemic and YS treated liver tissues were collected. The expression of RAE-1 and H60 genes was measured by RT-PCR and real-time quantitative PCR.Results: (1) YS treatment can effectively reduce the hige levels of ALT, AST, LDH and TNF-a in the sera caused by 90 min hepatic warm ischemia and 6 h of reperfusion. After YS treatment, the morphology and structure of vascular endothelium are relatively normal and the lymphocyte infiltration reduced.(2) YS treatment can effectively reduce the oxidative stress by elevating the SOD level and reducing the MDA lev更多还原