核因子-κB诱捕物ODN对大鼠移植肾的转染及对细胞因子基因表达的影响

Transfection of NF-κB decoy ODN in renal allografts and influence on the expression of chemokines of the renal grafts

目的 探讨核因子 -κB(NFκB)诱补物寡聚核苷酸 (ODN)对大鼠供肾的转染效率及对移植肾细胞因子基因表达影响。方法 建立大鼠移植肾模型 ,应用含有 1 μMLiposome/ODNEuro -Collins保存液(Ecs)灌注移植肾并低温缺氧保存 6h后移植到受体大鼠 ;2 4只大鼠分成 4组 :假手术组 (control组 ) ,ECs组 ,NFκB诱补物 (decoyODN)组 ,错配ODN(scrambledODN)组。应用荧光显微镜和多光子共聚焦显微镜观察转染有FITC标记的ODN移植肾 ,并观看其分布 ;用RT -PCR技术 ,检测术后移植肾中ICAM - 1、VCAM- 1、CINC的mRNA表达。结果 移植术后 1 5min ,FITC标记的decoyODN主要分布在移植肾的肾小球 ,并且有部分ODN进入细胞核 ;在术后 6h时 ,诱补物ODN主要分布在肾小球和近曲肾小管中 ,并且大部分进入细胞核。而不含有脂质体诱补物ODN复合物的Ecs保存的移植肾中则无荧光颗粒 ;与control组相比 ,ECs组移植肾 ,ICAM - 1、VCAM - 1、CINC的mRNA水平明显升高 (P <0 .0 5 ) ;与ECs组相比 ,NF -κB诱捕物ODN处理组移植肾的ICAM - 1、VCAM - 1、CINC的mRNA水平显著下降 (P <0 .0 5 ) ,而Scram .ODN处理组移植肾的ICAM - 1、VCAM - 1、CINC的mRNA表达则未见抑制 (P >0 .0 5 )。结论 诱捕物ODN脂质体复合物在ECs中及低温缺氧条件下可?

Objective To evaluate the transfection efficiency of nuclear factor κB(NFκB)decoy Oligodeoxynucletides (ODN)into donor kidney and the influnence of decoy ODN on the expression of chemokines of the renal grafts. Methods 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 1μM 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 1μM 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 gene expression of ICAM-1 VCAM-1 CINC in renal allografts were detected by RT-PCR. Result FITC-labeled ODN was mainly distributed in glomeruli of the transfected renal allograft at 15min posttransplantation, and mainly in glomeruli and the renal tubules at 6h posttransplantation. The most of ODN was transduced into nuclei at 6h posttransplantation. The fluroscence cann`t be seen in the ECs group. Compared to the control group, the gene expression of ICAM-1, VCAM-1,CINC was observably increased in ECs group (P>0.05); Conclusion Decoy ODN can be efficiently transfected into kidney using in vivo liposome method in ECs under hy pothermic and hypoxic condition; The renal allo-graft treated with decoy ODN can inhibit the gene expression of ICAM-1, VCAM-1, CINC.