阻断内毒素胞内信号转导通路对大鼠移植肝脏再灌注损伤的影响

The Influence with Block the Endotoxin Signal Transduction for Ischemia/Reperfusion Injury of Graft Liver in Rats.

目的探讨以白细胞介素-1受体相关激酶-4(IRAK-4)为靶点,阻断内毒素胞内信号转导后对大鼠移植肝脏再灌注损伤(I/RI)的影响并探索肝移植时可行的RNA干扰(RNAi)治疗途径。方法两袖套法建立SD大鼠同种异体原位肝移植模型,随机分为冷缺血转染组、活体转染组及对照组。冷缺血转染组于冷缺血期经门静脉灌注转染携带IRAK-4-shRNA的质粒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0min、60min及180min分为三个亚组,RT-PCR及Western-blot测定肝组织的IRAK-4mRNA和蛋白表达水平;ELISA法测定受体血清TNF-α含量。采用TUNEL法检测肝细胞凋亡状态,透射电镜观察肝组织超微结构的病理形态学变化。结果再灌注后冷缺血转染组的IRAK-4表达明显低于同时点的活体转染组及对照组(P<0.01);同时,肝细胞凋亡指数、血清TNF-α含量及肝细胞、血窦内皮细胞损伤程度也明显低于后者。结论以IRAK-4为靶点的冷缺血期shRNAs转染途径能有效阻断内毒素胞内信号转导,进而减轻肝移植时的I/RI程度。

Objective To explore the feasibility of interleukin 1 receptor associated kinase-4 （IRAK-4） as gene therapy target for liver ischemia/reperfusion injury （I/RI） and effective approach in vivo for short hairpin RNA （shRNA） interference used to gene therapy in liver graft hqappened. Methods Sprague-Dawley rats were randomly divided into three groups： the control group, the in vivo transfection group （IVT） and the cold ischemia transfection group （CIT）. Experiments of orthotopic liver transplantation were performed by two-cuff method. CIT were perfused with IRAK-4-shRNA plasmid （pSIIRAK-4） during cold ischemia phase, IVT received the equivalent volumes （2 mL） of pSIIRAK-4 after portal vein inosculated, and the control group leaved without any treatment. At 0 min, 60 min and 180 rain after reperfusion, the expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The serum TNF- α level was detected by ELISA. Liver histopathological changes and cell apoptosis were observed by electron microscope and TUNEL. Results After reperfusion, the expression of IRAK-4 were largely depressed in CIT than that of IVT and the control group （P〈0. 01）, and furthermore, the serum TNF-α level, proportion of hepatocyte apoptosis and severity of hepatocyte injury were also lower than the latter. Conclusion These results indicate that depression IRAK-4 expression with IRAK-4-shRNA through portal vein perfusion during cold i.schemia phase could effectively blunt graft hepatic I/RI.