| Objective: Liver transplantation has been the most effective therapy for various kinds of end-stage liver diseases.However, it has been reported that immunological rejection has happened to 60-65% of the patients who have received liver transplantation operation one year later. The main reason for that is the oxidative and inflammatory injury during liver cold preservation. At present more and more end-stage liver diseases patients need liver transplantation, the shortage of donor-liver becomes more seriously. So, how to more effectively protect the donor-liver and decrease the primary non-function (PNF) incidence abstract more and more attentions. Heme oxygenase is the rate-limiting enzyme in the catabolism of heme, a process that leads to formation of equimolar amounts of the bile pigment biliverdin, free iron, and carbon monoxide (CO), it has the following characters: anti-oxidation, anti-inflammation, anti-apoptosis, down regulation the expression of blood vessel endothelium adhesion molecule and decrease the injury of blood vessel and increase transplanted organs blood, the characters of HO-1 cause the extensive interestings of researchers. Protein transduction domains (PTDs) are peptides that can cross over the cell membrane, proteins or peptides can enter cells to exert their functions if they are linked to PTDs. HIV-1 trans-activator(TAT)is one of the most widely studied PTD. TAT can bring the proteins that fused to it into liver, kidney, myocardium and brain ,and so on . Some researches have confirmed that the fusion protein TAT-HO-1 can successively enter the pancreaticβTC-3 cells and strength their vitalities. Our prior researches show that the fusion protein TAT-HO-1 can enter the liver cell, and we come to a conclusion that TAT-HO-1can eliminate oxygen free radical, heighten the activity of SOD, lighten peroxidatic reaction. On the basis of prior study, we go on to make a research to investigate whether TAT-HO-1 would still transduce efficiently into the liver tissue cell and show its protective characters during cold preservation .Methods: The E.coli BL-21(DE3) containing TAT-HO-1- pET32a plasmid was cultured. The TAT-HO-1 protein was induced by IPTG and purified by Ni-NTA-agarose, then identified by Western blot analysis.Sixty male Sprague-Dawley rats weighing 250~300 g each were used for the study. Among the sixty rats, 12 ones were used to determine whether TAT-HO-1 would transduce into liver during cold storage. Livers were harvested as described by Kamada. After harvest, the livers were randomly divided into two groups according to the type of the preservation solution, Group HTK (Group C , livers were flushed and preserved with HTK solution at 4°C; Group protein (Group P , livers were flushed and preserved with HTK solution containing 50μg/ml TAT-HO-1 at 4°C). Liver specimens and preservation solution samples were collected at 0hr, 6 hr, 12 hr and 18 hr of cold storageImmunohistochemical analysis was performed to determine whether TAT-HO-1 would transduce into liver during cold storage. Selectra-E automatic biochemistry analyzer was employed to detect the concentrations of AST, ALT and LDH in perfusate. MDA level was detected by thiobarbituric-acid and SOD activity was detected by xanthine oxidase assay. TNF-αlevel was also detected by immunohistochemistry technique. Morphological changes were examined by light microscope and electron microscope.Results:1 The purified protein was confirmed to be TAT-HO-1,and the protein could enter the liver at the cold preservation.2 Enzymology results of the perfusateIn the two groups, with the prolongation of cold storage time, the levels of AST ,ALT and LDH were increasing, compared between times, there were significant differences (P < 0.05); At the time of 0h cold storage, there were no significant differences among the levels of AST,ALT and LDH (P >0.05), however at the time of 6h,12h and 18h,the levels of AST,ALT and LDH in the group protein were significantly lower than in the group HTK(P < 0.05). 3 The results of SOD activity, MDA and TNF-αcontents in liver tissueIn the two groups, with the prolongation of cold storage time, the contents of MDA and TNF-αincreased, compared between times, there were significant differences (P < 0.05); SOD activity decreased, compared between times, there were significant differences (P < 0.05);At the time of 0h cold preservation, there were no significant differences among SOD activity, MDA and TNF-αconcentrations (P>0.05),at the time of 6h,12h and 18h ,there were significant differences between both groups, the contents of MDA and TNF-αin the group protein were significantly lower than the group HTK, but the activity of SOD was significantly higher than the group HTK(P<0.05) .4 The results of morphological changesBy light microscope: At the time of 0h, the structure of hepatic sinusoid and liver cell cord were distinct, the outline of hepatocyte was clear and it was only slightly swollen, no necrosis was found .With the prolongation of cold storage time, the degree of injury aggravated gradually, the structure of hepatic sinusoid and liver cell cord disappeared time dependently, the hepatocyte swelled obviously, more necrosis were found. At the time of 6h,12h and 18h, the degree of injury in the group P was obviously lighter than group C.By the electron microscope: With the prolongation of cold storage time the mitochondrion swelled more obviously, mitochondrial crista became shorter and the number of it decreased from 50%~60% to 5%~10%, rough endoplasmic reticulum degranulation became more obviously. At the time of 6h, 12h and 18h, the degree of injury in the group P was obviously lighter than that in group C.Conclusions: TAT-HO-1 can transduce efficiently into rat livers during cold preservation.TAT-HO-1 added into HTK solution, shows better protective effect on liver against cold preservation injury than simple HTK solution. The mechanism is related to the anti- oxidation and anti-inflammation characters of HO-1.
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