活体/离体门静脉灌注转染IRAK-4-shRNA对大鼠移植肝脏再灌注损伤的影响

Effects of IRAK-4-shRNA perfused through portal vein in vivo or in vitro on ischemia/reperfusion injury of liver graft in rats

目的通过观察活体或冷保存期离体门静脉灌注供肝转染白细胞介素-1受体相关激酶-4(IRAK-4)特异短发夹 RNA(shRNA)对再灌注后受体 TNF-α产生的影响,判断以 IRAK-4为肝移植缺血再灌注损害(I/RI)治疗靶点的可行性并探索可行的 shRNA 治疗途径。方法雄性 SD 大鼠,随机分为冷缺血转染组、活体转染组、对照组,以两袖套法建立同种异体肝移植模型。冷缺血转染组于冷缺血期经门静脉灌注转染携带染 IRAK-4-shRNA 的转染质粒 pSIIRAK-4;活体转染组在门静脉袖套吻合完成后,经门静脉分支注入 pSIIRAK-4;对照组不予任何处理。按门静脉血流恢复后第0 min、60 min 及180 min 分为三个亚组,逆转录-聚合酶链式反应及蛋白免疫印记法测定肝组织的染IRAK-4 mRNA 和蛋白表达水平;酶连免疫吸附法检测肝组织 NF-κ B 活性及血清 TNF-α含量。结果再灌注后活体转染组、对照组的 IRAK-4蛋白与 mRNA 表达水平、NF-κB活性以及 TNF-α含量均高于冷缺血转染组(P<0.01);冷缺血转染组的 IRAK-4表达明显抑制,再灌注后各时点差异无显著性(P>0.05)。结论以 IRAK-4为靶点的冷缺血 shRNAs 转染途径能有效减轻肝移植时的 I/RI,但能否以 IRAK-4作为预防其他器官的 I/RI 的治疗靶点尚需深入研究。

Objective To investigate the changes of TNF-α in reperfusion host received graft liver transfected with short hairpin RNA （shRNA） targeting Interleukin 1 receptor associated kinase-4 （IRAK-4） gene through portal vein during cold ischemia period or in vivo after portal vein was inosculated to explore effective gene therapy approach and the possibility of IRAK-4 as genetherapy target for liver ischemia/reperfusion injury （I/RI）. Methods Sprague-Dawley rats were divided into three groups： the control group, the in vivo transfection group （IVT group） and the cold ischemia transfection group （CIT group）. The orthotopic liver transplantation were perfoumed by two-cuff method. The rats in CIT group were perfused with IRAK-4-shRNA plasmid （pSIIRAK-4） during cold ischemia period, those in IVT group received the equivalent volumes （2ml） of pSIIRAK-4 after portal vein inosculated and the control group leaved without any treatment. At 0, 60 and 180 min after reperfusion, the expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The activities of NF-κB and the serum TNF-α level were detected by ELISA. Results After reperfusion, all the indexes were significantly higher in IVT group and the control group than CIP group （P 〈 0.01）. However, the expression of IRAK-4 was significantly depressed in CIT group and the difference was not significant at different time phases after reperfusion （P 〉 0.05）. Conclusion The depression of IRAK-4 expression with IRAK-4-shRNA through portal vein perfusion during cold ischemia period can effectively protect against graft hepatic I/RI. However, the problem of whether IRAK-4 could be the ideal gene therapy target for I/RI of other organs needs to be further studied.