兔脑死亡状态下供体肝脏及肾脏质量评估研究

发布时间：2018-07-14 14:59

【摘要】：我国已经成为继美国之后的第二器官移植大国。移植受者的成活率也接近或达到国际先进水平。但是人体器官来源的匮乏成为严重制约移植事业发展的瓶颈。为与国际接轨,原国家卫生部和中国红十字会联合在全国16个省市自治区启动了心脏死亡器官捐献和移植试点工作。脑死亡供体将逐渐取代死囚,在未来两年内成为器官移植的主要来源。但由于大量临床研究报道,脑死亡供体与与活体亲属或尸体器官移植相比,近期或长期预后明显较差。前期研究报道,可能与脑死亡供体器官存在血流动力学改变、炎症因子释放、细胞凋亡、凝血因子消耗、内分泌及激素改变等病理生理改变有关,严重影响着脑死亡供体器官质量,这将成为制约脑死亡供体器官广泛应用的关键问题。目的：建立高度模拟临床、科学稳定的兔脑死亡动物模型。探讨脑死亡致兔肝脏及肾脏损伤的机制,寻找评估脑死亡供体肝脏及肾脏的灵敏指标,为改善脑死亡供体肝脏及肾脏质量提供方向。方法：(1)40只健康雄性12周龄新西兰兔随机分为假手术组与脑死亡组。各组再因脑死亡持续时间不同设置2、4、6和8h组(n=5)。假手术组依次行股动脉插管、气管插管、及颅骨钻孔置管术,不行颅内加压脑死亡术,持续至脑死亡组实验结束时问点；脑死亡组则依次行股动脉插管、气管插管、颅骨钻孔置管及缓慢颅内加压脑死亡术,呼吸机维持脑死亡状态到时间点。术中采用生物机能实验系统、动物呼吸机、智能恒温控制仪全程监控兔脑死亡状态建立及维持过程中的心率、呼吸波、平均动脉压以及脑电波。各组分别在脑死亡后2、4、6及8h收集动物血清、肝脏及肾脏组织标本。 (2)分别检测脑死亡组及假手术组脑死亡后2、4、6及8h各小组肝功能指标ALT和AST、肾功能指标BUN和Cr。采用光镜观察不同时间点肝脏及肾脏组织的形态学变化。同时采用ELISA试剂盒检测血清中IL-1β、IL-6、IL-8、TNF-α水平。免疫组化法检测肝脏及肾脏中炎症相关因子ICAM、HSP70。TUNEL法检测肝脏细胞及肾脏细胞的凋亡数目。以此初步评价脑死亡状态对供体肝脏及肾脏质量的影响。 (3)为进一步系统性评价脑死亡器官对这些因子或蛋白致脑死亡供体质量受损机理。我们借助蛋白质组学技术,即双向凝胶电泳技术、生物质谱技术及生物信息数据库分析技术对比筛选并鉴定脑死亡肝脏及肾脏组织与假手术组存在的差异蛋白,并采用western blot法验证了肝脏差异蛋白RUNX1的表达、免疫组化法验证肾脏差异蛋白PHB的差异。结果：(1)本文首次拟定的新西兰兔脑死亡模型建立标准,建立了全新的新西兰兔颅内缓慢加压脑死亡模型。建模过程中,脑死亡组颅内压升高引起动脉压骤升到峰值时明显高于假手术组(P0.05),但是脑死亡一段时间后,则急剧下降至正常水平以下(尸0.01)。但脑死亡建立尸过程中心率变化尚无显著差异,仅脑死亡2h后,脑死亡组心率明显低于假手术组(P0.01)。 (2)新西兰兔脑死亡后6h内肝脏功能及形态尚无明显变化,但脑死亡后8h之后,肝脏功能ALT、AST发生明显改变(P0.05),且肝细胞明显气球样变,肝窦受压,无明显肝索结构,汇管区有大量的淋巴细胞浸润,且伴有部分灶状坏死。脑死亡4h后虽然BUN值变化尚不明显(P0.05),但肾脏Cr显著升高(P0.05),肾小管细胞明显水肿、空泡变性也有所增加,部分近曲小管闭塞,炎症明显。IL-1β、IL-6、IL-8及TNF-a表达呈现逐渐上升趋势,且脑死亡后8h组各项指标明显高于假手术组(P0.05)。炎症相关因子ICAM逐渐上调(P0.05)、HSP70表达下调(P0.05)、凋亡细胞数目明显增加(P0.05) (3)蛋白质组学技术可以成功筛选并鉴定出脑死亡后肝脏差异较大的蛋白质有：线粒体醛脱氢酶,过氧化物酶6,3磷酸肌醇依赖性蛋白激酶1,3-巯基丙酮酸硫基转移酶,乙醇脱氢酶,二氢嘧啶酶相关蛋白4,Runt相关转录因子1,无机焦磷酸酶,谷氨酸-半胱氨酸连接酶的调节亚基及微粒细胞色素B5。且筛选出的差异蛋白质主要与细胞增殖与分化、物质代谢、解毒、抗氧化与氧化还原调节相关。筛选出的肾脏差异较大蛋白质有：抑制素,PRP38预mRNA处理因子38,钙调磷酸酶B1,V-type质子ATP酶亚基C1,NADH脱氢酶亚基10,过氧化物酶3,N-乙酰氨基半乳糖转移酶,膜粘连蛋白5,超氧化物歧化酶,细胞色素b-c1复合体亚单位1。主要与增殖与分化、信号转导、蛋白质的加工与修饰、电子传递链及氧化还原相关。其中,肝脏RUNX1表达随脑死亡时间延长表达下降(P0.05)。肾脏PHB表达随脑死亡时间延长表达逐渐上升(P0.05)。结论：本文建立的新西兰兔脑死亡模型是一种科学的、高度模拟临床的兔脑死亡模型。值得其他脑死亡动物实验推广及借鉴。兔脑死亡后早期,尽管肝肾功能尚未发生明显改变,但是已发生以炎症反应、细胞凋亡相关的复杂病理生理变化,影响着脑死亡兔肝脏及肾脏的质量。肝脏RUNX1表达可能成为兔脑死亡后肝脏质量评估的灵敏指标,但是其作用机制仍待进一步深入研究。肾脏PHB表达可能成为兔脑死亡后肾脏质量评估的灵敏分子标志物,为临床寻找新的方法改善脑死亡供体肾脏质量提供了新思路。
[Abstract]:China has become the second major organ transplant after the United States. The survival rate of transplant recipients is also close to or reached the international advanced level. However, the lack of human organ sources has become a serious bottleneck restricting the development of transplantation. In order to integrate with the international community, the former national Ministry of health and the Chinese Red tenth character association have joined up in the 16 provinces and autonomous regions of the country. Brain death donors will gradually replace death prisoners and become the main source of organ transplantation in the next two years. However, due to a large number of clinical studies, brain death donors have a poor short-term or long term prognosis compared with living relative or cadaver organ transplantation. Brain death donor organs have hemodynamic changes, inflammatory factors release, cell apoptosis, coagulation factor consumption, endocrine and hormonal changes and other pathophysiological changes, which seriously affect the quality of brain death donor organs, which will be the key problem that restricts the widespread use of brain death donor organs.
Objective: to establish a highly simulated clinical, scientific and stable animal model of rabbit brain death, to explore the mechanism of liver and kidney injury induced by brain death in rabbits, to find a sensitive index for evaluating the liver and kidney of brain dead donors, and to provide a direction for improving the liver and kidney quality of brain dead donors.
Methods: (1) 40 healthy male New Zealand rabbits were randomly divided into the sham operation group and the brain death group at 12 weeks old. Each group was set up 2,4,6 and 8h (n=5) because of the duration of brain death. The sham operation group was followed by femoral artery intubation, tracheal intubation, and cranioplasty, and no intracranial pressure brain death was performed, and continued to the end of the brain death group. In the brain death group, the femoral artery intubation, tracheal intubation, cranial drilling, and slow intracranial pressure brain death were performed in the brain death group. The ventilator maintained the brain death state to the time point. The biological function experiment system, the animal ventilator and the intelligent constant temperature controller were used to monitor the heart rate and respiration of the brain death state and maintenance of the rabbit brain during the operation. The mean arterial pressure and electroencephalogram were collected. Serum and liver and kidney tissue samples were collected from each group at 2,4,6 and 8h after brain death.
(2) the liver function indexes of 2,4,6 and 8h groups in the brain death group and the sham operation group were detected respectively, ALT and AST, and the renal function indexes BUN and Cr. were observed by light microscope to observe the morphological changes of liver and kidney tissues at different time points. The serum IL-1 beta, IL-6, IL-8, TNF- alpha levels were detected by ELISA kit. The liver and kidney were detected by immunohistochemical method. The number of apoptosis in liver cells and renal cells was detected by ICAM, HSP70.TUNEL, and the effect of brain death on the quality of donor liver and kidney was evaluated.
(3) in order to further systematically evaluate the mechanism of brain death organ damage to the brain death donor of these factors or proteins, we screened and identified the existence of brain dead liver and kidney tissue and sham operation group by means of proteomics, bi-directional gel electrophoresis, biological mass spectrometry and bioinformatics database analysis. Differential protein expression was detected by Western blot, and the differential protein RUNX1 expression in liver was verified by immunohistochemistry. The difference of PHB in renal differential protein was verified by immunohistochemistry.
Results: (1) a new model of New Zealand rabbit brain death model was established for the first time. A new model of brain death in New Zealand rabbits was established. In the process of modeling, the increase of intracranial pressure in the brain death group was significantly higher than that of the sham operation group (P0.05) when the intracranial pressure rose abruptly to the peak, but after a period of brain death, the brain death decreased to the highest level. There was no significant difference in the center rate of the brain death after the death of the brain (0.01). The heart rate of the brain death group was significantly lower than that of the sham operation group (P0.01) after the brain death was 2H.
(2) there was no obvious change in liver function and shape in 6h after the death of New Zealand rabbit, but after 8h, the liver function ALT, AST changed obviously (P0.05), and the liver cells were obviously balloon like, the hepatic sinusoids were pressed, no obvious hepatic cord structure, and there were a lot of infiltration of the lymphocytic cells in the sinks and some focal necrosis. The brain died after 4h, although BUN died of BUN The change of the value of the kidney was not obvious (P0.05), but the renal Cr increased significantly (P0.05), the renal tubule cells were obviously edema, the vacuolar degeneration was also increased, some of the proximal convoluted tubules were obliterated, and the expression of.IL-1 beta, IL-6, IL-8 and TNF-a in the inflammation was gradually rising, and the indexes of 8h group after brain death were obviously higher than that of the sham operation group (P0.05). The inflammation related factor ICAM was in the case of ICAM. Gradually increased (P0.05), HSP70 expression was downregulated (P0.05), and the number of apoptotic cells increased significantly (P0.05).
(3) proteomics techniques can be successfully screened and identified as proteins with large differences in the liver after brain death: mitochondrial aldehyde dehydrogenase, peroxidase 6,3 phosphoric acid kinase 1,3- mercapto pyruvate thiotransferase, ethanol dehydrogenase, two hydropyrimidase phase protein 4, Runt related transcription factor 1, inorganic pyrophosphatase, The regulated subunits of the glutamate cysteine ligase and the particle cytochrome B5. are mainly related to cell proliferation and differentiation, substance metabolism, detoxification, antioxidation and redox regulation. The larger proteins of the kidney are inhibin, PRP38 premRNA treatment factor 38, calcineurin B1, V-type substance. The subunit ATP subunit C1, NADH dehydrogenase subunit 10, peroxidase 3, N- acetaminophen galactotransferase, membrane adhesion protein 5, superoxide dismutase, and cytochrome b-c1 complex subunit 1. are mainly related to proliferation and differentiation, signal transduction, protein processing and modification, electron transfer chain and redox related. The expression of RUNX1 in the liver is associated with the brain The prolongation of death time decreased (P0.05). The expression of PHB in kidney increased gradually with the prolongation of brain death time (P0.05).
Conclusion: the New Zealand rabbit brain death model established in this paper is a scientific, highly simulated rabbit brain death model, which is worthy of the promotion and reference of other brain dead animal experiments.
In the early days after the death of the rabbit brain, the liver and kidney function had not changed significantly, but the complicated pathophysiological changes related to the inflammatory reaction and apoptosis have affected the quality of the liver and kidney of the brain dead rabbits.
The expression of RUNX1 in the liver may be a sensitive index for evaluating the quality of the liver after the death of the rabbit, but the mechanism of its action remains to be further studied. The expression of PHB in the kidney may be a sensitive molecular marker for the evaluation of kidney quality after the death of the rabbit brain, which provides a new way of thinking for the clinical search for a new method to improve the quality of the renal death donor kidney.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R699.2;R657.3

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