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[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R699.2;R657.3

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