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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、空白腺病毒对照组(空白对照组)、生理盐水对照组(NS对照组)。于肝移植前48 h分别用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)均显著低于空白对照组和NS对照组(P<0.01),而空白对照组、NS对照组比较无统计学差异(P>0.05);恢复灌注12 h Ad-RPS3-shRNA组各检测指标与3 h变化趋势相同。结论沉默供体肝脏内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。
Objective To silence the expression of ribosomal protein S3 (RPS3) gene in donor liver by RNA interference (RNAi) technique and observe its effect on nuclear factor-kappa B (NF-κB) Protective effect of early postischemic reperfusion injury. Methods The RPS3 gene-specific short hairpin RNA (shRNA) expression plasmid containing adenovirus (Ad) as carrier was constructed and divided into three groups according to random number table: Ad-RPS3-shRNA group, blank adenovirus control Group (blank control group), saline control group (NS control group). 48 h before liver transplantation, three groups of donor rats were treated with Ad-RPS3-shRNA particles, blank adenovirus vector and normal saline respectively. Blood and liver tissues were collected for the detection of serum aminotransferase, plasma inflammatory cytokines, histopathological changes of liver tissue, mRNA of inflammatory cytokines and NF-κB activity and expression level in liver tissue extracts. Results The levels of serum ALT (725.9 ± 67.3) U / L, AST (863.7 ± 72.7) U / L, and the level of plasma TNF-α in the Ad-RPS3-shRNA group were 223.7 ± 16.8 ng / L, the level of IL-1β was (242.5 ± 18.7) ng / L, the level of ICAM-1 was 258.4 ± 24.9 ng / L and the score of Suzuki was 3.72 ± 0.31. , IL-1β (0.217 ± 0.028), ICAM-1 (0.226 ± 0.017), RPS3 (0.094 ± 0.007), liver tissue NF- κB protein level (0.216 ± 0.022) and their activity (92.4 ± 9.5) (P <0.01), but there was no significant difference between the blank control group and the NS control group (P> 0.05). The detection indexes of Ad-RPS3-shRNA group and the change trend of 3 h after reperfusion for 12 h the same. Conclusion Silencing the RPS3 gene in the donor liver can significantly decrease the activity and expression of NF-κB in liver cells, thereby reducing the expression of inflammatory cytokines and effectively reducing the early post-transplantation ischemia-reperfusion injury.