| Background: Our country is one of countries with the higher morbidity and mortality of liver ailment in the world. Liver transplantation is developing quickly and the number of patients been performed liver transplantation is increasing in our country. Liver dysfunction or failure is a significant clinical problem after liver transplantation. The dramatic organ shortage forces lots of marginal grafts been used. These grafts have a higher susceptibility to ischemia-reperfusion injury and a higher possibility of primarily liver dysfunction after transplantation, which directly limits the developing of clinic liver transplantation. How to avoids or limits its happen is the focal point of the liver transplantation research.Some research indicated, in the processes of the ischemia-reperfusion injury of liver transplantation, the activation of NF-κB which leading into the expression of lots of inflammation cytokines plays a very important role. So we can suppose inhibit the activation or expression of NF-κB maybe have a use to lessen the ischemia-reperfusion injury in these process. The technique of RNA interference has a dominantly use of silencing special gene expression and an extensively utilize prospect in organ transplantation. Effective gene therapy requires a reliable gene transfer tools to efficiently insert target genes into the cells of the grafts. Adenovirus vectors have been favored because of their propensity to infect hepatic cells and their ability to infect proliferation and nonproliferation cells.PART I:Optimize the parameters for transfecting siRNA into cultivated rat BRL cells.Objective: To optimize the parameters for transfecting siRNA into cultivated rat BRL cells by RNAi-mate in vitro.Methods: Two different density of RNAi-mate were mixed with four different density of fluorescin - labeled siRNA respectively, and then eight mixtures were got. Each of the mixtures was used to transfect BRL cells.24 hours after transfection; cell count was made by fluorescent microscope to determine the percentage of the cells been transfected under each condition. The cell viability was calculated by using MTT assay at the same time.Results: In the case of 1.5ug siRNA plused with 3.0ul RNAi-mate, we found the transfection efficiency was 89%, the cell survival rate was 84%, and it was the best parameter.Conclusions: In our experiment, when transfectting BRL cells, the best ratio of siRNA: RNAi-mate was 1:2.PART II: The gene silence effect of the siRNA on the NF-κB p65 expression of the BRL cellsObjective: To choose the NF-κB p65 siRNA with the highest gene silence efficacy from the 4 pairs of siRNA.Methods: We designed 4 pairs of siRNA targeting rat NF-κB p65 mRNA. Combined with control group there were 7 experiment groups altogether. Then we transfected those siRNA into the cultivated rat BRL cells use the way we conformed in part 1. The NF-κB p65 gene expression effectiveness was tested by reverse transcription-PCR and Western blotting.Results: Though the suppressing degree was different, each one of those 4 pairs of siRNA could suppress the NF-κB p65 gene expression from the mRNA and protein level. The second siRNA was the most efficient one and the NF-κB p65 mRNA expression was decreased 90%, the protein expression was decreased 80% compared with the blank group.Conclusions: Our experiment confirmed, each one of those 4 pairs of siRNA has the function of gene silence and the second one was the most efficient one.PART III: Construct a recombinant Ad containing NF-κB p65 specific siRNA expression cassette and its amplification, purifying and titrating.Objective:To construct a recombinant Ad containing NF-κB p65 specific siRNA expression cassette.Methods:The NF-κB p65 specific siRNA expression cassette was cloned behind the sequence of H1-RNA promoter, and the positive clones containing target gene were selected and appraised. A recombinant adenovirus was produced by a double-recombination event between cotransformed adenoviral backbone plasmid pAdEasy-1 and a linearized shuttle vector. Positive clones were selected and confirmed. Then they were linearized and transferred into the 293A cell to amplification, and then were purified by density gradient ultracentrifuge and titrated. The desired Ad vectors were transfected into the BRL cells to detect the gene silence efficiency.Results:Both the plasmid pShuttle-H1-p65 and the recombinant Ad were confirmed correct. The recombinant Ad were generated in a high titer which reached 3.0×1010 pfu/ml, and could efficiently knock down the NF-κB p65 expression in vitro.Conclusion: we successfully constructed the recombinant Ad which containing NF-κB p65 specific siRNA expression cassette.PART IV: The establishment of a model of rat orthotopic liver transplantationObjective:To establish a reliable animal model of rat orthotopic liver transplantation with modified cuff technique.Methods:140 cases of rat orthotopic liver transplantation were performed by using the two-cuffed technique with some modification in graft harvesting, the anastomoses of suprahepatic vena cava and the perioperative treatment. Results:Of the last 30 cases, the mean time of anhepatic period was 12min, The 24 hours and 2 week survival rates were 93% and 90% respectively.Conclusions: The modified two-cuffed technique is convenient and easy to repeat. It can enhance the stability and survival rate of rat orthotopic liver transplantation models. Skilled surgical techniques and shortening the anhepatic phase of recipient as much as possible are the keys to animal survival.PART V: Adenovirus expressing shRNA targeting p65 have the protective effect against the ischemia-reperfusion injury of rat liver graftsObjective:To test the Gene silencing efficacy of Ad-shRNA-p65 in vivo and its potential protective effect against the ischemia-reperfusion injury of liver grafts.Methods: The donor rats was infused with the Ad- shRNA- p65 in a titer of 3×109 pfu via portal vein. 36 hours later, half of the rats were executed to detect the items as below: Serum ALT,AST,LDH; the liver pathology changes; the expression of NF-κB p65 mRNA and protein in the liver. The other donor rats were re-laparotomized to harvest the grafts and the grafts were preserved for 4 hours in Ringer's lactate solution. Then the orthotopic rat liver transplantation (OLT) was performed using the modified two-cuffed technique described in the partⅣ. Six hours after transplantation, the expression of NF-κB p65 mRNA and protein and TNF-α,MIP-2,ICAM-1mRNA in the liver were tested; the bile volume, content of MDA in the liver and serum ALT,AST,LDH were measured, while NF-κB p65 and TNF-αcontent were estimated by immunohistochemistry and apoptosis was analyzed by TUNEL.Results:The mRNA and protein expression of NF-κB p65 were decreased in the donor rats which infused in Ad- shRNA- p65. and infusing Ad would induce the heighten of serum ALT,AST,LDH. Six hours after reperfusion, in the experiment group, the bile volume was increased, the expression of NF-κB p65 mRNA and protein , TNF-α,MIP-2,ICAM-1mRNA in the liver, the content of MDA in the live, the level of ALT,AST,LDH in serum were all decreased, compared with control groups. The immunohistochemistry showed that lower expression of NF-κB p65 and TNF-αin the liver tissue of the experiment group and TUNEL showed less apoptosis cells .Conclusions: Administered via portal vein, the recombinant adenovirus expressing shRNA targeting p65 could obviously silencing the NF-κB p65 expression in rat liver. Infuse in Ad itself maybe had a little degree of liver damage. Donor adenovirus-mediated gene transfer of Ad- shRNA- p65 could decrease subsequent ischemia-reperfusion injury in rat liver transplantation. One of its mechanism maybe explained as down-expression of NF-κB p65 will lead into the inhibit expression of TNF-α,MIP-2,ICAM-1, and thus reduce the leukocytes infiltrating the liver.
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