慢病毒介导siRNA对大鼠脊髓组织细胞外信号调节激酶1/2及核因子E2相关因子2基因的影响

Effect of lentivirus-mediated si RNA on extracellular-signal-regulated kinase 1/2 and nuclear factor-E2-related factor 2 in spinal cord tissue of rats

目的评价慢病毒介导细胞外信号调节激酶1/2(extracellular-signal-regulated kinase 1/2,ERK1/2)-si RNA及核因子(nuclear factor,NF)E2相关因子2(NF-E2-related factor 2,Nrf2)-si RNA进入大鼠局部脊髓组织的转染效果。方法采用NYU撞击法构建SD大鼠脊髓损伤模型(模型组),使用微量注射器将含荧光慢病毒载体(LV-Nrf2-RNAi和LV-ERK1/2-RNAi)稀释液注射至其脊髓T10水平背侧深约0.8 mm处,荧光显微镜下观察不同时间点(12 h、24 h、72 h、7 d及14 d)转染效果。以只切除椎板,不打击脊髓的大鼠作为假手术组,在模型组和假手术组中同时设转染组和未转染组。取大鼠脊髓组织,采用RT-PCR及Western blot法检测转染后7 d时ERK1/2及Nrf2基因m RNA转录及蛋白表达水平。结果与假手术组相比,模型组大鼠脊髓组织在慢病毒转染后各时间点均有较强的荧光表达,病毒转染7 d以内,荧光强度与时间呈正相关,在转染7 d后达到高峰。转染7 d后,假手术组与模型组中ERK1/2及Nrf2基因m RNA转录及蛋白表达水平均较未转染组明显降低(P均<0.05)。结论慢病毒介导si RNA转染大鼠脊髓组织,能够有效沉默ERK1/2及Nrf2基因的表达。

Objective To evaluate the lentivirus-mediated transfection of extracellular-signal-regulated kinase 1 / 2（ERK1 / 2）-si RNA and nuclear factor（NF）-E2-related factor 2（Nrf2）-si RNA in spinal cord tissue of rats. Methods SD rat model of spinal injury was established by using NYU impactor（model group）. The rats receiving laminectomy without weight drop were served as sham operation group. The rats in both groups were injected with diluted solution containing fluorescence lentiviral vector into the dorsal column of the spinal cord at T10 of rats in two groups by microsyringe, at a depth of 0. 8 mm, using those untransfected as control, and observed for transfection efficacy at various time points（12 h,24 h, 72 h, 7 d and 14 d） by fluorescent microscopy. The expressions of ERK1 / 2 and Nrf2 m RNAs and proteins in rat spinal cord tissue 7 d after transfection were determined by RT-PCR and Western blot. Results Compared with that in sham operation group, strong fluorescence was observed in spinal cord tissue of rats in model group at various time points after transfection with lentivirus, of which the intensity was in a time-dependent mode within 7 d and reached the peak value 7 d after transfection. However, both m RNA and protein expression levels of ERK1 / 2 and Nrf2 in sham operation and model groups 7 d after transfection were lower than those without transfection（each P〈 0. 05）. Conclusion Lentivirus-mediated RNA interference effectively silenced the expressions of ERK1 / 2 and Nrf2 genes in spinal cord tissues of rats.