脑死亡状态下大鼠肝组织中基因表达变化及肝损伤防护的实验研究

发布时间：2018-06-07 16:57

本文选题：脑死亡 + 基因表达　；参考：《郑州大学》2016年博士论文

【摘要】：目前,肝脏移植的供肝主要来自脑死亡患者的捐献。然而,实验和临床研究发现:与活体供肝相比,脑死亡供肝移植术后的缺血再灌注损伤重、急性排斥反应发生率高,出现初始肝功能不良和原发性移植物无功能的风险大。因此,国内外学者建立了大量动物脑死亡模型来研究脑死亡状态对潜在供体器官的影响以及可能的干预措施。脑死亡是指“包括脑干在内的全脑功能不可逆性的丧失”,可以引起机体复杂的病理生理变化,主要表现为血流动力紊乱、激素水平改变、炎症和免疫系统被激活、补体和凝血系统被激活。这些不利因素导致线粒体和内质网应激细胞凋亡通路被激活,使细胞凋亡增加,严重影响了脑死亡供肝的质量。但是,脑死亡状态下肝损伤的具体分子机制仍不清楚。因此,研究脑死亡状态下肝脏组织中基因的变化,可以为减轻脑死亡状态下肝损伤提供潜在的干预靶基因。血红素加氧酶1(heme oxygenase 1,HO-1)又称为热休克蛋白32(heat shock protein,HSP32),是降解血红素的限速酶,具有抗炎和抗凋亡的作用。研究发现,脑死亡状态下肾脏中HO-1和HSP70高表达。钴原卟啉(cobalt protoporphyrin,CoPP)预处理脑死亡供体诱导HO-1高表达,减轻移植肾中炎性细胞浸润,并可以将脑死亡供肾移植后3个月的移植物存活率从20%显著提高至50%。脑死亡状态是否促进肝脏中热休克蛋白的表达,ho-1诱导剂-copp是否能减轻脑死亡状态下肝脏损伤,其分子作用机制是什么至今还不清楚。本课题拟从以下三个部分进行研究,建立大鼠脑死亡模型,分析脑死亡状态下大鼠肝组织中基因表达情况,探讨copp对脑死亡状态下大鼠肝损伤的分子作用机制,以期为提高脑死亡供肝质量提供新的思路和方法,从而为临床肝移植提供更多可用的脑死亡供体。第一部分:缓慢间断颅内加压法建立大鼠脑死亡模型目的:为研究脑死亡状态对肝脏质量的影响,建立一种稳定、可靠的大鼠脑死亡模型。方法:采用sd大鼠,颅骨钻孔在硬膜外腔放置3ffogarty球囊导管,通过间断缓慢加压法诱导大鼠脑死亡。以自主呼吸停止、脑电波静息、脑干反射消失和深昏迷作为脑死亡判定标准。尾静脉补羟乙基淀粉溶液维持脑死亡6h期间平均动脉压大于80mmhg。脑死亡组20只大鼠;假手术对照组(n=10)不行球囊加压,其余操作同脑死亡组。监测大鼠的动脉血压、心率、呼气末co2、补液量和尿量,检测血清alt和ast水平,观察肝组织病理变化。结果:大鼠脑死亡模型的成功率为90%,加压诱导前准备时间为70±10min,脑死亡加压诱导时间为34±6min,加压球囊所需的液体量为136±24μl。脑死亡组大鼠的羟乙基淀粉输入量为36.46±17.85ml,尿量为32.77±18.44ml,血清中alt和ast分别为70.17±15.87u/l、201±23.31u/l,均明显高于假手术对照(均p0.05)。光学显微镜下观察可见,假手术对照组大鼠的肝脏组织形态正常;脑死亡组的大鼠肝脏呈现明显的淤血和水肿,肝索增宽,肝窦变窄,存在散在的点状坏死。第二部分:脑死亡状态下大鼠肝组织中基因表达变化目的:分析脑死亡状态下大鼠肝组织中基因表达变化,为揭示脑死亡状态下肝损伤的分子机制提供实验依据。方法:分别在大鼠脑死亡后0h、1h、2h、4h、6h留取标本,假手术对照组大鼠也在相对应的时间点留取标本,每个时间点6只大鼠。用agilent大鼠全基因组表达谱芯片分析脑死亡6h及假手术对照的大鼠肝脏组织中41012个基因的表达情况。qpcr检测大鼠脑死亡后0h、1h、2h、4h、6h肝脏中ho-1、hsp27、hsp70、mcl-1、bcl-2、bnip3、caspase-3、hif-1α、hif-2α、hif-3α、arnt和glut-1的表达情况。westernblotting和免疫组化检测大鼠脑死亡后肝脏中ho-1蛋白表达情况。结果:与假手术对照组比,脑死亡6h大鼠肝脏组织中有149个基因表达上调超过10倍,有136个基因表达下调超过10倍。脑死亡后0h、1h、2h、4h、6h的大鼠肝脏中ho-1的mrna和蛋白表达水平明显上调,ho-1蛋白表达在胞浆,在肝小叶中央静脉周围的肝脏细胞高表达。bnip3和caspase-3的mrna表达水平在脑死亡后0h、2h、4h、6h大鼠肝脏中明显上调。第三部分:copp减轻大鼠脑死亡状态下肝损伤的分子作用机制目的:探讨ho-1诱导剂-copp对大鼠脑死亡状态下肝损伤的作用及其分子机制。方法:将大鼠随机分成4组,每组6只。脑死亡(b)组:大鼠脑死亡维持6h;假手术(s)组:不加压球囊诱导脑死亡,其余操作同脑死亡组;copp预处理脑死亡(cb)组:术前24h腹腔注射copp5mg/kg,其余操作同脑死亡组;copp预处理假手术(cs)组:术前24h腹腔注射copp5mg/kg,其余操作同假手术组。全自动标准生化分析仪检测血清中alt和ast的水平。westernblotting检测肝脏中bcl-2、bax、mcl-1、chop、caspase-12、cytosoliccytochromec和cleaved-caspase-3蛋白的表达。tunel检测大鼠肝脏中细胞凋亡情况。结果:copp预处理显著诱导大鼠肝脏中ho-1蛋白高表达。与脑死亡组比,copp预处理脑死亡组大鼠的血清alt和ast水平明显降低,肝脏中bcl-2和mcl-1蛋白表达明显上调,而bax、chop、caspase-12、cytosoliccytochromec和cleaved-caspase-3蛋白表达明显下调,肝脏中凋亡细胞明显减少。结论:1.建立了稳定、可靠、重复性好的大鼠脑死亡模型,为进一步研究脑死亡状态对供体器官的影响提供了基础。2.首次全面分析了脑死亡状态下大鼠肝脏组织中基因的表达,为减轻脑死亡状态下肝脏损伤提供了新的干预靶基因;脑死亡后肝脏组织中HO-1表达上调,可能与脑死亡相关应激启动的保护性机制有关。3.HO-1诱导剂-CoPP可以显著上调HO-1蛋白表达,并通过调控线粒体和内质网凋亡途径中关键蛋白,减轻脑死亡大鼠肝脏中细胞凋亡。HO-1有望成为提高脑死亡供肝质量的潜在干预靶点。
[Abstract]:At present, donor liver transplantation is mainly from the donation of brain death patients. However, experimental and clinical studies have found that compared with living donor liver donor liver transplantation, cerebral death has a high incidence of ischemia-reperfusion injury, high incidence of acute rejection, high risk of initial liver dysfunction and primary graft without function. A large number of animal brain death models have been established to study the effects of brain death on the potential donor organs and possible interventions. Brain death refers to the loss of the irreversible function of the whole brain, including the brain stem, which can cause complex pathophysiological changes in the body, mainly characterized by disturbance of blood flow, changes in hormone levels, and inflammation. The disease and immune system are activated and the complement and coagulation system are activated. These adverse factors lead to the activation of the apoptosis pathway of mitochondria and endoplasmic reticulum stress cells, increase the apoptosis of cells and seriously affect the quality of brain death donor liver. However, the specific sub mechanism of liver injury in brain death is still unclear. Therefore, the study of brain death is in the state of brain death. The changes in the gene in the liver can provide potential intervention targets for reducing the liver injury in the brain. Heme oxygenase 1 (heme oxygenase 1, HO-1), also known as heat shock protein 32 (heat shock protein, HSP32), is a speed limiting enzyme that degrades heme, and has the effect of anti-inflammatory and anti apoptosis. HO-1 and HSP70 were highly expressed in the viscera. Cobalt protoporphyrin (CoPP) pretreated the brain death donor to induce the high expression of HO-1, alleviated the infiltration of inflammatory cells in the transplanted kidney, and could increase the graft survival rate from 20% to the death state of the brain 3 months after the transplantation of the brain to the death state of 50%. to promote the expression of heat shock protein in the liver. It is not clear what the molecular mechanism of the HO-1 inducer -copp can reduce the liver injury in the brain death state. This topic is to establish the rat brain death model from the following three parts, analyze the gene expression in the rat liver tissue under the brain death, and discuss the copp damage to the rat liver in the brain death state. Molecular mechanism of action, in order to provide new ideas and methods for improving the quality of brain death donor liver, provides more available brain death donors for clinical liver transplantation. Part 1: the aim of establishing rat brain death model with slow intermittent intracranial pressure method is to study the effect of brain death on the liver quality and establish a stable and reliable large scale. Method: the rat brain death model was used in SD rats. The 3ffogarty balloon catheter was placed in the epidural cavity in the skull hole, and the brain death was induced by intermittent slow compression. The brain death was determined by the stop breathing, the brain wave resting, the brain stem reflex disappearing and deep coma as the criteria for brain death. The tail vein supplementation hydroxyethyl starch solution maintained the brain death period of 6h. The mean arterial pressure was greater than that of 20 rats in the 80mmhg. brain death group; the sham operation control group (n=10) had no balloon pressure, the rest of the operation was with the brain death group. The blood pressure, heart rate, CO2, rehydration and urine volume of the rats were monitored, the levels of serum ALT and AST were detected, and the changes of liver tissue were observed. The results were as follows: the success rate of the rat brain death model was 90% The preparation time was 70 + 10min before induction, and the pressure induction time of brain death was 34 + 6min. The amount of liquid needed in the pressure balloon was 136 + 24 Mu L. brain death group, the hydroxyethyl starch input was 36.46 + 17.85ml, the urine volume was 32.77 + 18.44ml, and the serum ALT and ast were 70.17 + 15.87u/l and 201 + 23.31u/l respectively. All were significantly higher than those of the sham operation control (p0.0, all p0.0). 5). It was observed under the optical microscope that the liver tissue of the rats in the sham operation control group was normal, and the liver of the rats in the brain death group showed obvious congestion and edema, the widened hepatic cord, the narrowing of the hepatic sinus and the scattered necrosis. The second part: the change of gene expression in the rat liver tissue under the brain death: the analysis of the large brain death state. The changes in gene expression in rat liver tissue provide experimental basis for revealing the molecular mechanism of liver injury in the brain dead state. Methods: 0h, 1H, 2h, 4h, 6h were left after the death of the rat brain, and the rats in the sham operation control group were also left at the corresponding time points, and each time point was 6 rats. The whole genome expression chip of Agilent rats was used. Analysis of the expression of 41012 genes in the liver tissues of the brain dead 6h and the sham operation control rats.Qpcr detection of 0h, 1H, 2h, 4h, 6h liver HO-1, HSP27, HSP70, Mcl-1, Bcl-2, HSP70, Mcl-1,.Qpcr and immunohistochemical detection of the liver after the death of the rat brain after brain death The expression of medium HO-1 protein. Results: compared with the sham control group, the expression of 149 genes in the liver tissue of 6h rats was up to 10 times higher than that of the sham control group, and the expression of 136 genes was down 10 times. The mRNA and protein expression of HO-1 in 0h, 1H, 2h, 4h, 6h of the rat after brain death was up-regulated, HO-1 protein expressed in the cytoplasm and in the hepatic lobule. The expression level of high expression of.Bnip3 and Caspase-3 in the liver cells around the central vein was obviously up-regulated in the liver of 0h, 2h, 4h, 6h rats after brain death. The third part: copp alleviated the molecular mechanism of the liver injury in the brain death of rats: the effect of HO-1 inducer -copp on the liver injury in the rat brain and its molecular mechanism Method: the rats were randomly divided into 4 groups, 6 rats in each group. Brain death (b) group: rat brain death maintained 6h; sham operation (s) group: non pressurized balloon induced brain death, the rest of the same brain death group; copp pretreated brain death (CB) group: preoperative 24h intraperitoneal injection of copp5mg/kg, the rest of the same brain death group; copp preconditioning pseudopero operation (CS) group: preoperative 24h abdominal abdominal cavity Injection of copp5mg/kg, the rest of the operation with the sham operation group. Test the level of ALT and AST in serum by automatic standard biochemical analyzer to detect the expression of Bcl-2, Bax, Mcl-1, chop, caspase-12, cytosoliccytochromec and cleaved-caspase-3 protein in the liver. The high expression of HO-1 protein in the rat liver was induced. Compared with the brain death group, the level of serum ALT and AST in the rats with copp pretreated brain death group decreased significantly, the expression of Bcl-2 and Mcl-1 in the liver was obviously up, while the expression of Bax, chop, caspase-12, cytosoliccytochromec and cleaved-caspase-3 egg white was obviously down, and the apoptotic cells in the liver decreased significantly. Conclusion: 1. a stable, reliable and reproducible rat brain death model was established, which provided a basic.2. for the first time to analyze the expression of gene in the rat liver tissue in the brain dead state for the first time, and to provide a new target gene for reducing the liver injury in the brain death state. The up-regulated expression of HO-1 in the post liver tissue may be related to the protective mechanism of brain death related stress..3.HO-1 inducer -CoPP can significantly up-regulate the expression of HO-1 protein, and by regulating the key proteins in the apoptosis pathway of mitochondria and endoplasmic reticulum, the decrease of apoptosis.HO-1 in the liver of brain death rats is expected to improve the liver quality of brain death. Potential targets for quantitative intervention.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R741;R657.3

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