Study On NF-κ B Decoy ODN Reduce The Initiating Injury Of Renal Allografts In The Rat

Objective: 1. To investigate the effect of H/R(hypoxia/reoxygenation) on the activity of NF- K B and the expression of adhesion molecular and chemokines of ECV-304; 2. To assess efficiency of FITC-labeled ODN mediated by liposome in the ECV-304; 3. To evaluate the influence of decoy ODN on the activity of NF- K B and the activation of adhesion molecular and chemokines in the ECV-304; 4. To establish the rat kidney transplantation model and observe the effect of the preservation of the kidney in ECs containing decoy ODN/Liposome complexes; 5. To evaluate the transfection efficiency of decoy ODN using liposome method into donor kidney under hypothermic and hypoxia condition and the affection of decoy ODN in the early injury of the renal grafts.Methods: 1. The ECV-304 monolayers were subjected to hypoxia for 6h in ECs at 4 癈 followed by Oh 1h 2h 4h 6h 12h reoxygenation, respectively. The DNA binding abitility of NF- K B was measured by EMSA.The gene expression of IC AM1, VCAM-1, P-selectin, IL-8, MCP-1 were detected by RT-PCR. 2. ECV-304 monolayers were transfected with Liposome/ODN complexes containing 0.5? M,0.75 祏 M, 1.0 ?M, 1.25 ?M ODN respectively in ECs at 4 癈 and then stored for 2. 4.6 and 8 hours respectively under hypoxia. The distribution of FITC-labeled ODN in ECV-304 cells were observed by fluorescence microscope. The transfection efficiency and MFI were evaluated by flow cytometry. 3.The DNA binding abitility of NF- K B in ECV-304 cells which were transfected by decoy ODN and subjected to 6h hypoxia followed by 4h reoxygenation were measured by EMSA. Meanwhilethe gene expression of adhesive molecules and chemokines were detected by RT-PCR. 4. 25 adult male SD rats and Wistar rats, haevested donor organs include arteria abdominalis, left kidney, left kidney vessels, left ureter, vesica urinarya and a part of bladder. Titanium clips technique and/or continuous overhand suture were applied in anastomosis for kidney transplantation, and titanium clips technique was used in anastomosis for ureter and bladder. 5.The rats were divided into 4 groups. Control group: the Wistar rats underwent the sham operation and the kidney was harvested after 6h. ECs roup: The donor kidney was stored for 6h under of hypothermic and hypoxia condition in ECs without ODN/Liposome complexes, then the donor kidney was transplanted into a SD recipient. The allograft was harvested at 6h posttransplantation. Decoy ODN group: The donor kidney was stored for 6h in ECs containing 1 u M Liposome/decoy ODN complexes under hypothermic and hypoxia condition, then the donor kidney was transplanted into a SD recipient. The allograft was harvested at 6h posttransplantation. Scrambled ODN group: The donor kidney was stored for 6h in ECs containing 1 u M Liposome/scrambled ODN complexes under hypothermal and hypoxia condition, then the donor kidney was transplanted into a SD recipient, and at 6h posttransplantation the allograft was harvested. The distribution of FITC-labeled ODN in renal tissue were observed by fluorescence microscope and TCS SP2 Confocal microscope .The effect of NF- K B decoy ODN on the activity of NF- KB in renal allografts were measured by EMS A; The gene expression of ICAM-1 VCAM-1 MCP-1 RANTES in renal allografts were detected by RT-PCR; Renal allograft structure was assessed by light microscopy; The count and distribution of EDl-postive cells in renal allograft were evaluated by immunohistological study.Result.1. The activation of NF-B in ECV-304 at Ih after reoxygenation starts rising and gets to peak at 4h, then begin to drop at 12h; and the gene expression of ICAM-1, VCAM-1, P-selectin, IL-8 and MCP-1 were significantly upregulated in ECs group compared to the control group.2. MFI of Liposome/ODN group was 1072.71 + 33.14, and was 91.1 folds of the naked ODN group. The transfection efficiency of Liposome/ ODN group was 93.06+2.60%, and was 71.5 folds of the naked ODN group; Intensive fluorescence was seen in nuclei from the Liposome/ODN group, however, it was rarely seen in nuclei from the...