1.The Protective Effect Of Ischemic Postconditioning On Rat Liver Grafts 2.The In Vitro Research Of Immune Tolerance Induced By Silencing CD40 Production In Dendritic Cells Derived From Rat Bone Marrow

Part 1: The protective effect of ischemic postconditioning on rat liver graftsHepatic ischemia-reperfusion (IR) injury is a common pathological process of traumatic surgical diseases in the liver, such as severe liver trauma, extensive hepatic lobus excision, liver transplantation, shock and infection. Despite impressive improvement in preservation techniques and liver transplantation outcomes during the last decade, IR injury associated with liver transplantation is still an unresolved problem in the clinical practice, resulting in initial poor function and primary non-function of liver allograft. Therefore, it is of great clinical interest to elucidate the underlying pathomechanisms and to develop protective strategies. Although the specific mechanisms are still unclear, it likely various mechanisms have been implicated in liver IR injury, including reactive oxygen species (ROS), lipid peroxidation, cytokine production, alterations in calcium homeostasis, activation of Kupffer cells, mitochondrial dysfunction and so on. Although seemingly counterintuitive, ischemic preconditioning (IPC) has been proved to significantly attenuate IR injury in a variety of mammalian tissues including heart, kidney, skeletal muscle, small bowel, and liver. Despite the powerful protective effects of IPC, the clinical application of this phenomenon has been rather disappointing, mainly because it must be instituted before the ischemic event. In this regard, the newly described phenomenon of ischemic postconditioning (IPostC), which consists of several very short circles of ischemia and reperfusion applied immediately at the onset of reperfusion, offers a far more attractive and amenable approach to myocardial protection. In the present study, we established orthotopic liver transplantation (OLT) model of rats and observed the effect of IPostC on liver grafts, to determine the role of IPostC in protecting liver grafts from prolonged IR injury after OLT and try to elucidate the related mechanisms.Objective: The purpose of this study was to establish liver transplantation model of rats with same strain, investigate the protective effect of IPostC on ischemia reperfusion injury of rat liver grafts and to ascertain if neutrophil recruitment and nitric oxide (NO) production were implicated in the process.Methods: Male Spraque-Dawley rats, weighing 280-320g, were used as donors and recipients and randomly divided into four groups. IPostC was achieved by several intermittent interruptions of blood flow in the early phase of reperfusion followed by a persistent reperfusion. (1) In sham-operated (SO) group, after laparotomy, only the left phrenic vein and the right suprarenal vein were ligated and the hepatic artery was freed by ligating and dividing, without OLT; (2) In ischemia-reperfusion (IR) group, standard OLT was performed; (3) In ischemic postconditioning 1 (IPostC1) group, same as IR group, but immediately at the onset of reperfusion, reflow was initiated with 30 s of full portal vein flow, followed by 30 s of re-occlusion with non-trauma mini artery clamp, repeated twice more for a total of three circles; (4) In ischemic postconditioning 2 (IPostC2) group, same as IPostC1 group, but six circles of 30 s reperfusion separated by 30 s re-occlusion described above were imposed immediately after donor liver was implanted. The recipients were sacrificed for sample collection of blood and hepatic tissue after 6 hours of reperfusion. Plasma samples were collected from inferior vena cava, and separated by centrifugation, and median lobe of the liver was carefully excised and stored at -80℃for analysis. The severity of hepatocellular injury was determined by serum levels of hepatic enzymes. Concentration of malondialdehyde (MDA), activities of antioxidant enzymes and neutrophilic infiltration in hepatic tissue, serum levels of tumor necrosis factor (TNF)-αand NO contents were compared between groups. The proportion of necrotic areas to the total hepatic areas in each section was determined using HPIAS-100 image analytical system.Results: (1) donator operation time: 30.1±3.26min, donator liver set up time: 17.3±2.14min, receptor operation time: 46.4±3.60min, abhepatic phase: 18.5±1.62min, operation achievement ratio: 75%, primary causes of operation failure were hemorrhage and thrombogenesis. (2) Compared with SO group, ALT, AST and LDH in IR group were significantly elevated after OLT, the values of ALT, AST and LDH were significantly suppressed when the liver grafts were treated with postconditioning; Compared with IPostC 1 group, IPostC 2 group didn't have further effect. (3) The concentration of MDA in hepatic tissue was rapidly elevated after reperfusion in IR group, while the activities of SOD and GSH-PX were significantly reduced. Compared with IR group, the concentration of MDA in IPostC1 or IPostC2 group was markedly reduced and the activities of both antioxidant enzymes were significantly enhanced. (4) In IR group, the level of MPO in hepatic tissue was rapidly elevated after reperfusion. Compared with IR group, the levels of MPO were significantly reduced in IPostC1 and IPostC2 group. No statistical differences were observed between two IPostC groups. (5) Compared with SO group, the level of TNF-αin serum increased significantly in IR group. In two IPostC groups, the increases were reduced. (6) Compared with SO group, the content of NO was elevated in IR group and two IPostC groups, moreover the increases were prominent in two IPostC groups. (7) Sections of IR group presented disorderly liver sinusoids, hepatocellular necrosis and massive infiltration of neutrophil, IPostC group showed good preservation of lobular architecture, with less neutrophil infiltration and minimal hemorrhage.Conclusions: (1) In the present study, we successfully established liver transplantation model of rats with same strain; (2) The results indicate that IPostC has a protective effect against IR injury after OLT in rats. (3)IPostC maybe apply its protective effect through attenuating neutrophil accumulation and elevating NO production. (4) Simply extending the postconditioning algorithm in ischemic postconditioning could not have further protective effect. (5)The intervention of ischemic postconditioning is very simple. Unlike preconditioning, postconditioning theoretically allows unrestricted application in the clinical settings, and thus has direct clinical application value.Part 2: The research of immune tolerance induced by silencing CD40 production in dendritic cells derived from rat's bone marrowGraft rejective reaction is an unresolved problem for organ transplantation. Immunosuppressive drug can not always effectively control the reject reaction. Moreover, immunosuppressive drugs have side effects and long-term use may induce opportunistic infection and tumor. The idealistic solution is to induce the immune tolerance of recipient to donator. DC is the most powerful specific antigen presenting cell(APC), can activate na?ve T cells and prime primary immune response, regulate the balance between immune activation and immune tolerance. CD40 is the acceptor of CD40L, expressed on many kinds of cells, such as DC, B lymphocyte, macrophage, endotheliocyte, and so on. It has recently been discovered that short sequences of RNA that are 21 nt in length (known as small interfering RNA or siRNA) can bypass the broad suppression of the IFN response and can lead to the specific degradation of cognate mRNA. This process, known as RNA interference (RNAi), is immensely specific, since a single substitution in the 21-nt sequence can abrogate its effects, and extremely efficient, since the siRNA is incorporated into an enzymatic complex that conducts multiple rounds of target mRNA degradation. As such, RNAi provides a powerful tool for inhibiting endogenous gene expression and could provide a means to effectively modulate immune responses.Objective: The purpose of this study was to establish a method of inducing DC from rat bone marrow cells in vitro, construct and identify the eukaryotic expression plasmids encoding two siRNAs of rat CD40 gene, observe the change of phenotype and immunologic fuction in DCs transfected with the eukaryotic expression plasmids.Methods: Two sequences corresponding to the rat CD40 gene were designed on Ambion's Web. The two complementary oligonucleotide strands of DNA fragments were synthesized by chemosynthesis. After annealing of the complementary strands, the DNA fragments were connected to the polyclone sites of pSilencer 4.1-CMV neo vector. Followed by transformation, amplification and plasmid extraction in E.coli, the recombinant plasmids were identified by agarose gel electrophoresis by means of cutting with BamHⅠand HindⅢand by DNA sequence analysis. The rat bone marrow cells were collected and cultured in vitro under the condition of rrGM-CSF and rrIL-4. After 2 weeks, the morphological character of DCs was observed under light microscope and scanning electron microscope. Expression of MHC-Ⅱ, CD80 and CD86 were detected by flow cytometry. The ability to stimulate allogenic T cells of the cultured DCs was detected by mixed lymphocyte reaction. On day 6, the recombinant plasmids were transfected into DCs. The expression of CD80, CD86 and MHC-Ⅱwas detected by flow cytometry. RT-PCR analysis and Western-blot were used to detect CD40 mRNA transcription and expression. ELISA was used to detected IL-10 and IL-12 levels in supertant of DC culture. MLR was used to detect the allostimulatory ability of transfected DCs.Results: (1) The connections between the DNA fragments encoding CD40-targeted siRNA and the pSilencer 4.1-CMV neo vector were correct, as confirmed by agarose gel electrophoresis and DNA sequence analysis. (2) DC displayed typical morpholory with elongated dendritic processes viewed by inversion microscope and scanning electron microscope. DCs at day 6 revealed immature phenotype, including MHC-Ⅱ29.1%, CD80 21.9%, CD86 25.2%. DCs at day 12 showed higher expression of MHC-Ⅱ(76.2%), CD80(21.9%) and CD86(25.2%), and they also revealed higher stimulatory capacity of allogenic T cells than DCs of day 6. (3) The result of RT-PCR and Western-blot indicated that the transcription of CD40 gene was suppressed by RNAi. (4)Flow Cytometry assay showed that transfection of DC with pS-CD40A did not affects the phenotype. (5)ELISA results showed that IL-10 production in DC treated with pS-CD40A was significantly up-regulated and the IL-12 production was significantly down-regulated. (6)The MLR results showed that the allostimulatory ability of the transfected DC was inhibited.Conclusion: (1) The two recombinant plasmids expressing siRNA of rat CD40 gene were successfully constructed. (2) Cultured with attendance of rrGM-CSF and rrIL-4, matured DC could be generated from rat bone marrow cells after 2 weeks. (3) pS-CD40A could effectively silence CD40 gene expression on DCs, the allostimulatory ability of the transfected DC was inhibited. These data demonstrated that RNA interference is a potent and specific tool for modulating DC-mediated immune responses.