| Ischemia/reperfusion injury(IRI) and acute rejection(AR) remain two important factors affecting kidney allograft after transplantation. IRI is inherent in transplantation because every donor vascularized organ by definition suffers varied degree ischemia and reperfusion. IRI may lead to increased immunogenicity of the graft, resulting in acceleration of Ag-specific processes such as acute rejection episodes and decreased long-term graft survival due to the progression of chronic-type rejection. There is a growing recognition now that long-term graft failure is at least in part influenced by the consequence of transplantation-related IRI. With current immunosuppressive protocols resulting in significant reduction in the rate of acute rejection, increasing attention has been directed toward Ag-independent factors influencing allograft survival. Hence, it is important to develop novel strategies to limit or prevent the graft injury, which in turn should ultimately result in the improvement of the long-term allograft outcome. Recently years, with the application of gene therapy techniques to transplantation, local cytoprotection and/or immunosupression in graft may be obtained, and gene therapy has been a promising way to prevent renal from IRI.Nitric oxide synthase (NOS) is an important mediator in both normal and pathologic renal environments. Inducible NOS (iNOS) has been found in segments of the renal tubule, glomerulus, and interlobular and arcuate arteries of normal rat kidneys . Recently, it has been suggested that NO produced from iNOS is detrimental in I/R, and that excessive amounts of NO produced I /R association with increased activity of iNOS can be cytotoxic.RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing mechanism, which is triggered by double-stranded RNA and causes degradation of mRNAs homologous in sequence to the dsRNA. Recently, RNA silencing has attracted much attention as a tool in biology. A unifying feature of all RNA silencing phenomena is the production of small RNAs that act as specificity determinants for downregulating gene expression.Lentiviruses such as HIV are members of the family of retroviruses. They are able to integrate into the genome of nondividing cells, which makes them an attractive tool for gene transfer in the central nervous system. An important aspect of lentivectors is that they can be used for gene transfer in ES cells and preimplantation embryos, which is the basis of lentiviral transgenesis. In this study, we combined lentiviral transgenesis with RNAi to evaluate the therapeutic potential of anti-iNOS siRNAs in rat.Part 1 Construction of a recombinant Lentiviruse carrying iNOS siRNAs gene Find the rat gene sequence from the Genbank and design the sequence of siRNA by the manufacturer. select effctive sequence of siRNA and transfer the sequences into cells effectively. Contrivances four target sequence, first we do the experiment in in vitro:uses the plasmid Construct Clon carrying agent, shRNAs were synthesized using the AmpliScribe, then link the target sequence and Clon agent. The approach described here enabled us to select highly effective rat siRNA. Amplificat the Clon sequence by polymerase chain reaction and ensure correct of the target sequence.Minimal packaging plasmids of Lentiviruse: Lentiviruse packag, Lentiviruse transfect 293T cell, viral replication, concentrate the viral, lentivector transfer plasmids were cloned with a variety of RNA export/transport elements to determine their ability to generate lentivectors titers, through the Western Blot and real-time PCR detect the efficacy of RNA interference. At last, select the best one that its efficacy is biggest and to pack it. Concentrate lentivectors titers make it can be use the sequentia experiment.Part 2 The protection of recombinant Lentiviruse carrying iNOS siRNAs gene against renal ischemia-reperfusion in ratsIn order to study the protection against renal ischemia/reperfusion with the recombinant adenovirus, the model of kidney ischemia-reperfusion was established with Sprague-Dawley rats. In brief, the right kidney was cut off firstly, and the left kidney was flushed via abdominal aorta with 0～4℃HC-A organ storage solution. 1 hour late, the left kidney was reperfused。The blood samples were collected at2d,3d,5d,7d,14d and 21d after the kidney reperfusion for the BUN,Cr assaying, meanwhile, the left kidney was cut off at the same time after the kidney reperfusion and prepared for further analyses. In the therapy team, every left kidney was perfused and preserved with Lentiviruse carrying iNOS siRNAs after flushed with 0～4℃HC-A organ storage solution via renal artery. The rats in control groups were perfused with 0.9% normal saline solution slide chemical methods. The kidney samples of rats were harvested for assaying of histology, immunohistochemistry. The results showed that perfusion and preservation with Lentiviruse via renal aorta could transfect renal cells of rats effectively. By immunohistochemical and enzymatic activity of iNOS assay, we found that the rats treated with Lentiviruse carrying iNOS siRNAs degraded iNOS mRNA in kidneys at a high level. According to this, the level of BUN,Cr in serum were decreased, and the change in histology (HE staining and transmission electron microscoping) was ameliorated.Conclusion:We draw a conclusion that recombinant Lentiviruse carrying iNOS siRNAs gene can be introduced into kidney during cold preservation effectively in IRI model. The target organ can degrade iNOS mRNA in kidneys, which can attenuate the kidney ischemia/reperfusion injury in rats. These findings provide some experimental evidences that gene delivery of sequences encoding graft cytoprotection can be full considered for clinical application in renal transplantation.
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