沉默供体肝脏内核糖体蛋白S3基因减轻大鼠移植肝脏缺血再灌注损伤.docVIP

沉默供体肝脏内核糖体蛋白S3基因减轻大鼠移植肝脏缺血再灌注损伤 陈 新，成名翔，龚建平，陈 勇，涂 兵（400010 重庆，重庆医科大学附属第二医院肝胆外科） ［摘要］ 目的 采用 RNA干扰技术沉默供体肝脏内核糖体蛋白S3(ribosomal protein S3, RPS3)基因的表达，观察其对核转录因子kappa B(nuclear factor-kappa B,NF-κB)的影响以及对肝移植后早期缺血再灌注损伤的保护作用。方法 构建以腺病毒(Ad)为载体的RPS3基因特异性短发夹RNA(shRNA)表达颗粒，按随机数字表法将实验大鼠分为3组:Ad-RPS3-shRNA组(Ad-blank-control组)(NS-control组)Ad-RPS3-shRNA颗粒、空白腺病毒载体、生理盐水处理3组供体大鼠。收集血液及肝脏组织检测血清转氨酶、血浆炎症因子水平、肝脏组织病理改变、炎症因子mRNA、肝组织提取物中NF-κB活性和表达水平指标的变化。结果 肝移植后恢复灌注3 h Ad-RPS3-shRNA组大鼠血清ALT水平(725.9±67.3)U/L、AST水平(863.7±72.7)U/L，血浆TNF-α水平(223.7±16.8)ng/L、IL-1β水平(242.5±18.7)ng/L、ICAM-1水平(258.4±24.9)ng/L，Suzuki评分(3.72±0.31)，肝组织mRNA水平：TNF-α(0.252±0.026)、IL-1β(0.217 ± 0.028)、ICAM-1(0.226 ± 0.017)、RPS3(0.094±0.007)，肝组织NF-κB蛋白水平(0.216±0.022)及其活性(92.4±9.5)均显著低于Ad-blan组NS-control组(P0.01)，而Ad-blan组NS-control组无差异(P0.05)；恢复灌注12 h Ad-RPS3-shRNA组各检测指标与3 h变化趋势相同。RPS3基因可显著降低肝脏细胞NF-κB活性和表达量,从而降低炎症因子表达，有效减轻肝移植后早期缺血再灌注损伤。 ［关键词］ RNAi；NF-κB； RPS3 in donor lives impairs ischemia reperfusion injury following orthotopic liver transplantation in rats Chen Xin, Cheng Mingxian, Gong Jianpin, Chen Yon, Tu Bing (Department of Hepatobiliary Surgery, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China) ［Abstract］ Objective Using the RNAi technique to silence donor liver ribosome protein S3 (RPS3), observe the influences to nuclear factor-kappa B (NF-κB) and early ischemia/reperfusion injury (I/RI) after liver transplantation in rats. Methods Construction of recombinant Adenovirus-RPS3-shRNA granules and the rats were divided into Ad-RPS3-shRNA, Ad-blank-control, NS-control groups. Ad-RPS3-shRNA, blank adenovirus granules and normal saline were injected into prospective donor rats before the donor liver was harvested 48h. The liver tissue and blood samples were harvested after surgery protocols. Liver function, Plasma cytokines and Histopathological were assessed; meanwhile real time polymerase chain reaction, electrophoretic mobility shift assay and western blotting were performed to asses