| Since the 1990s, the clinical liver transplantation has made considerable progress, the resource of donors is building up, according to the materials statistics, the number of patients who waited for liver transplantation has increased to 128% only during the recent 3 years in US. The legislation of"brain death"impulsed enormously organ transplantation in each country. The donator can take from the patients of"brain death"who are under the favourable breath and cycle state. However, the donator is insufficient relatively which still puzzles the patients who is waiting for liver transplantation. Although partial living liver transplantation, split liver transplantation and diferent spieces of liver transplantation may increase the resource of partial donator, but it has its applied local. How to appraise, utilize and improve non-heart-beating donors (NHBD) objectively, which has become investigative hot spot today. Quibus NHBD has become the humen beings'investigative focus again, which can increase the resource of donors[1]. According to the above, this project established rat NHBD model with sudden cardiac arrest and rat NHBD with slow cardiac arrest, through biochemistry, cytology and pathology method to examine and compared the difference of graft viabilities. In the experiment of liver transplanation from NHBD, cardiac arrest was induced by the injection of KCL in some studies. In this model, there were no conditions such as low arterial blood pressure, low concentration of oxygen content, or acidosis that were always recognized in clinical course from the brain death to the cardiac arrest. At the same time, we chose to use the agonal NHBD model induced by cessation of the respiration. In this study we clarify the difference between the sudden cardiac arrest model by injection of KCL and the slow (ie, agonal cardiac arrest) model by opening the chest wall using rat. Results in our study clearly indicated the agonal cardiac arrest model had low PaO2, severe deterioration of mitochondrial viability, and slight damage of parenchymal hepatocytes compared with those in the sudden cardiac arrest model. The mechanism and degree of deteriorarion in the sudden cardiac arrest model were considerably different from the agonal NHBD. Compared with ideal donors of keeping good breath circulation, NHBD model must meet serious warm ischemia coup. In this experiment, postop ALT, LDH value and the result of liver pathology reflected that the damage of warm ischemia to hepatic cell will be more and more serious following the asystole to extend. This kind of the detriment will show rapidly at the beginning of reperfusion. The aggravation mechanism of warm ischemia liver transplantation has already proved gradually, IDEM effect/ reperfusion injury was one of the important reasons why donors were nonfunctioning. And it was important decisive factor of succeeding in liver transplantation.In the early ischemical reperfusion injury ( in 2 hours after reperfusion ) mainly was caused by dysbolism and oxidative stress: anaerobic glycolysis and ATP decomposition, acidism in the cells, ph turn lower, mitochondria membrane permeability was changed. Oxygen deficiency, IDEM effect and oxidative stress made mitochondria membrane lipid peroxidation, mitochondria membrane permeability was changed, transmembrane potential was decreased, the lever of energy composition was cut down, bioblast swelled and disaggregated, it caused that cells function lose. Therefore, the change of mitochondria membrane permeability is one of the key steps of causing cell death. Reactive oxygen species(ROS), hypsi-concentration Ca2+ and oxidant is the important factor of causing the change of mitochondria membrane permeability.In the recent 10 years, the domestic and foreign scholars have conducted the massive research to the liver preserved damage mechanism and methods, the majority of scholars believed, although the ultra oxygen ion/free radical is not only one etiological factor of ischemical reperfusion injury, but it is also very considerable component element. The former research demonstrated that the ultra oxygen ion/free radical mediated ischemical reperfusion injury mainly generated the period of reperfusion, but this year it was discovered that transplant organ also has ultra oxygen ion/free radical scavenger was fore treated or added it to preservative fluid, it may obviously reduce the sensibility of liver to ischemical reperfusion injury. The commonly used oxygen free radical scavenger such as erythrocuprein, purinase, glutathione and so on. Although peroxydase has important sense to many kinds of morbid precaution and management as a result of free radical damage caused. But because natural peroxydase as anti- oxidized medicine has all sorts of shortcomings, prepared with the mimic enzyme have the for medicinal purposes value the peroxide enzyme simulation to become the new research direction. According to the structure of enzyme simulant, may divide into simple organic compound, peptide stimulant and protein simulant. Compare Peptide mat with simple organic compound, because it has the same constitutional unit with protein- amino acids, so it has some merits such as toxicity slightly, the vigor is high, composition is easy. Compared with protein simulant, it has some merits such as molecular mass is diminutive, the constancy is higher. At the same time modern peptide synthesis technology causes any peptide sequential that it is possible.Antiscorbic acid peroxydase (APX) is a kind of peroxydase that is based on ferroheme. It utilizes antiscorbic acid (AsA) as electron donor to eliminate H2O2 in vivo. Fe3+ in the ferroheme porphyrin ring is important group of its binding site. Fe3+ ionic electronic structure, oxidation state and porphyrin ring to extra ligand's affinity determines mimic enzyme activeness. In the natural peroxydase, histidine residue, configuration and a polypeptide chain pathognostic three-dimension structure on a polypeptide chain, which sublimates its activity greatly. MP-8, MP-9 and MP-11, that is, octapeptide, nonapeptide and hendecapeptide covalent linkage's microperoxidase , which were obtained by the cytochrome C hydrolysis. But because they were not easy in vitro to synthesize has limited the application. Because alkenyl on the ferroheme was easily oxidated by perhydride, accordingly it degraded constancy to perhydride. So we deployed Subloed A ferroheme to substitute ferroheme, which synthesized Subloed A ferroheme- brachypeptid chemical compound mimic enzyme DhHP-6 and discovered its activity had achieved above MP-11 95%, and has the better anti- oxidized activeness.This project using DhHP-6 as ultra oxygen ion/free radical mundificant affects rats liver transplantation donor model, and we has conducted the experimental study in the cellular level, subcellsular level, molecular level and pathohistology. The ferroheme hexapeptide (DhHP-6) is mimic enzyme of antiscorbic acid peroxydase (APX), the enzyme catalyzes H2O2 decompose to H2O under the antiscorbic acid existence. Peroxydase simulant DhHP-6 is prepared by chemical synthetic process, this enzyme has the higher oxidized activity.This research by H2O2 simulation peroxidation damage established liver cell peroxidation damage model. Some experiments proved H2O2 may cause the dose dependent cell death, the change of cell persistence is thoroughly personalization that H2O2 creates the frozen-in damage to the cell. This experiment discovered that after dealing with H2O2 30min, the cell persistence starts to reduce and the low density H2O2 (100μmol·L-1) may show conspicuous the cell damaging effect following temporal extension. Firstly, cell function was changed; and then the damage accumulates to the certain degree has triggered cell organic lesion and frozen-in damage.This experiment uses cultivanting liver cell of neonate rats as the damage object, it has consummate metabolism function and has avoided nerrohumor, excitatory autacoid level all over the body and part different type intercellular interaction. And it surmounted individual difference utmostly, has the very strong specificity. The H2O2 density approaches to ischemic reperfusion when ROS produces, creates the process of damage is similar to pathology process that ROS in vivo causes the liver cell damage. Therefore this model simulation is very strong; Moreover, this model's controllability is strong, and it is easy to duplicate. Raise neonate rats liver peroxidation damage model in vitro to carry on energometry, whose result showed, the H2O2 damage group liver cell's vigor remarkably is lower than various dosage DhHP-6 protection group, indicating DhHP-6 may effectively reduce the hydrogen peroxide to the liver cell's damage function and protect cell vigor. Because the number of the cells is invariable in the certain time, and MTT reflects change of the cell bioblast dehydrogenase activeness. Bioblast suffers injury, dehydrogenase activeness is lower and biocatalyst outleakage all can reduce Formazan production.In the experiment using perhydride mimic enzyme DhHP-6 as essential component prepared parenchymatous organ preservative fluid to compare UW preservative fluid. Liver transplantation donor model's influence: NHBD model group liver SOD activeness drops obviously, and MDA content increase obviously, indicating when rats liver ischemical reperfusion injury following free radical's accumulation and antioxidase activeness's reducing.The DhHP-6 preservative fluid can reduce obviously liver MDA content. This experiment indicated, when liver ischemical reperfusion injury, not only does the ultra oxygen ion/oxygen free radical production increase, but also the ultra oxygen ion/oxygen free radical's cleaning capability is degraded. Thus, it creates the ultra oxygen ion/free radical which in the liver organization produces massively accumulation, causes liver organizational major injury. DhHP-6 can reduce obviously liver ischemical reperfusion injury rat blood serum liver MDA content and elevate liver SOD activity, cue it possibly through suppress lipid peroxidation process. Lipin peroxidation is spell membrane damage or disorganization is one of cell damage important mechanisms. And simultaneously it enhances endogenous antioxidase activity to reduce the ultra oxygen ion/free radical to the liver damage. Indicating perhydride mimic enzyme DhHP-6 catalysises hydrogen dioxide to form water. Above the results possibly becomes the commonly used ultra oxygen ion/oxygen free radical elimination medicinal preparation regarding development oxide compound simulation enzyme DhHP-6 to reduce the donor to ischemical reperfusion injury. Finally it applies to DhHP-6 to clinically, has the vital significance.This research main outcome includes: We established NHBD models with sudden cardiac arrest and slow cardiac arrest, detected and compared their donors'activity. Therefore, we can conclude that in the experiment al study of NHBD transplantation the agonal NHBD model should be used. Compared with ideal donors of keeping good breath circulation, NHBD model must meet serious warm ischemia coup. Therefore, giving the objective evaluation and reasonable option to liver of NHBD play a important role on elevating this kind of liver posttransplantation persistence. In this experiment, which synthesized Subloed A ferroheme- brachypeptid chemical compound mimic enzyme DhHP-6 and discovered its activity had achieved above MP-11 95%, and has the better anti- oxidized activeness. For continuing to seek the activity higher mimic enzyme build the foundation; This research by H2O2 simulation peroxidation damage established liver cell peroxidation damage model. Raise neonate rats liver peroxidation damage model in vitro to carry on energometry, whose result showed, DhHP-6 may effectively reduce the hydrogen peroxide to the liver cell's damage function and protect cell vigor. Therefore, preliminary screening DhHP-6 is used as ultra oxygen ion/free radical scavenger, but this year it was discovered that transplant organ also has ultra oxygen ion/free radical scavenger was fore treated or added it to preservative fluid. Using DhHP-6 as the principal constituent compounds the substantive internal organs preservation fluid, and with cytology method confirmed DhHP-6 regard the ultra oxygen ion/oxygen free radical mediated ischemical reperfusion injury protection function in rat liver transplantation modle , which are new opinions of this research.By Medline confirmes, this research is about DhHP-6 to the rat ischemical reperfusion injury protection function result in rat liver transplantation modle, this belongs to creative study all over the world. About DhHP-6 regard the ultra oxygen ion/oxygen free radical mediated ischemical reperfusion injury protection function in rat liver transplantation modle, which is written by our research group. DhHP-6 as one kind of new ultra oxygen ion/free radical scavenger can reduce sensibility of live to ischemical reperfusion injury and at the same time it has no cytotoxicity; DhHP-6 is the principal constituent preservative fluid become a new strategy of elevating liver transplantation donator activity. And we discusses thoroughly DhHP-6 prevention and treats the ultra oxygen ion/free radical mediated ischemical reperfusion injury mechanism, which conduces to take high-quailty donors. The DhHP-6 screening is helpful to obviate and treat oxygen free radical mediated ischemical reperfusion injury to provide the new peroxydase.
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