Nuclear Factor-Kappa B In Coordination With CTLA4-Ig To Prevent Acute Rejection After Liver Transplantation In Rat

Objective This study was conducted (1) to isolate and culture kupffer cells (KCs) in rat, observe the activation of KCs with or without transduced nuclear factor-kappa B (NF-κB) decoy oligodeoxyribonucleotide (decoy ODN) under stimulation of lipopolysaccharide (LPS), and detect suppression effect of NF-κB decoy ODN on KCs activation; (2) to build experimental model of orthotopic liver transplantation in rat (ROLT), summarize operative skills and technique, observe the development process and basic pathophysiologic changes of acute rejection (AR) following allotransplantation from Lewis (LEW) to Brown Norway (BN) rat; (3) to observe employing cytotoxic T lymphocyte associated antigen 4-Ig (CTLA4-Ig) alone or in combination with NF-κB decoy ODN to prevent acute rejection of allograft in rat, and elucidate the molecular mechanism of the combination approach. Methods (1) KCs were isolated with in situ collagenase perfusion method and were randomly divided into three groups: control group, LPS stimulation group (1mg/L LPS), and NF-κB decoy group, in which the KCs were transduced with fluorescein isothiocyanate (FITC) labeled NF-κB decoy ODN (4μg/1x105KCs) prior to LPS stimulation. The transfection effect was detected by fluorescence microscope. Following 6 h of LPS stimulus, the phagocytosis function of KCs was detected by India ink test. The translocation of NF-κB was detected by immunocytochemical staining, NF-κB activity was assayed by electrophoretic mobility shift assay (EMSA), CD80 mRNA expression in KCs was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the production of TNF-αand IL-6 in supernatant were measured by ELISA. (2) The animal model of orthotopic liver transplantation in rat was performed with two-cuff technique, of which suprahepatic vena cava (SHVC) was anastomosed with continuous suture, portal vein (PV) and infrahepatic vena cava (IHVC) were anastomosed with cuff method, and the bile duct was reconstructed by interior stent. The rats were randomly divided into three groups: control group, isotransplantation group (LEW-LEW), and allotransplantation group (LEW-BN). Recipients were sacrificed on 3 d, 5 d, 7 d, and 10 d postoperatively and liver tissues and blood samples were collected. Recipient survival rate, histopathological and ultrastructural characteristics were observed, Plasm levels of alanine aminotransferase (ALT), total bilirubin (TBIL), and albumin (Alb) were measured with automatic biochemical analyser. IL-2 content in serum was assayed by ELISA. (3) Allotransplantation from LEW to BN rat was performed with two-cuff technique, and recipients were randomly divided into four groups: acute rejection group, in which recipients received no treatment; CTLA4-Ig group in which CTLA4-Ig (0.25 mg/kg body weight) were injected intraperitoneally daily for 7 days starting on day 0 postoperatively; NF-κB decoy group, in which 120μg decoy ODN was injected into donor via caudal vein 2 day prior to operation; combination group (0.25 mg/kg CTLA4-Ig in coordination with 120μg decoy ODN). Recipients were sacrificed on 7 d postoperatively, liver tissues and blood samples were collected. KCs in liver graft were isolated, the efficiency of decoy ODN transduction was detected by fluorescence microscope. NF-kB activity was measured with enzyme linked immunosorbent assay (ELISA). CD80, CD40, and CD54 mRNA expression were measured with RT-PCR. TNF-α and IFN-γ mRNA in liver graft were detected with in situ hybridization.Apoptosis cells in graft and spleen were detected with terminal-deoxynucleotidyl mediated nick end labeling (TUNEL). Recipients survival rate, histopathological and ultrastructural characteristics were observed, serum levels of ALT, TBIL, and Alb were also measured. All values are expressed as mean±SEM. one-way ANOVA was used to determine the significance of differences in multiple comparisons. A value of P <0.05 was considered significant. Result (1) The NF-κB decoy ODN can be efficiently transduced into KCs mediated by Lipofectamine, and reached 85% tr...