慢病毒介导白介素-1β siRNA对脊髓钝挫伤大鼠运动功能的影响

The effect of lentivirus-mediated interleukin-1β siRNA on motor function in spinal cord contused rats

目的:观察白介素-1β(IL-1β)小干扰RNA(si RNA)对脊髓钝挫伤(spinal cord contusion,SCC)大鼠运动功能的影响,并探讨其相关机制。方法:98只雌性SD大鼠随机分为4组:假手术组(Sham组)、单纯SCC组(SCC组)、空载体组(Vector组)和慢病毒干扰组(IL-1βSH组)。IL-1βSH组和空载体组术前48h在拟损伤脊髓节段局部注射IL-1βsi RNA或空载体。Sham组大鼠只剥离椎板不实施SCC,其余3组大鼠均造成T10SCC。每组各取大鼠5只,于术前当天以及术后1d、3d、5d、7d、14d采用Basso Beattie&Bresnahan(BBB)评分评估大鼠后肢运功功能。Sham组术后28d处死大鼠,取T10段脊髓,用实时荧光定量聚合酶链式反应技术(qRT-PCR)检测IL-1β和γ-突触核蛋白(SNCG)mRNA相对表达水平。SCC组于术后6h、12h、1d、3d、5d、7d、14d取损伤段脊髓,检测IL-β和SNCG mRNA相对表达水平。Vector组和IL-1βSH组在术后7d取损伤段脊髓检测SNCG mRNA相对表达水平;在术后3d、7d、28d行心脏灌注、固定损伤脊髓,用免疫组织化学染色技术观察SNCG的免疫阳性细胞计数,并测定平均积分光密度(IOD)值。应用GeneMANIA分析IL-1β和SNCG的理论关系。结果:Sham组各时间点BBB评分均为21分,SCC后,SCC组评分各时间点均显著低于Sham组(P<0.05),IL-1βSH组评分在3d、5d、7d、14d时均显著高于Vector组(P<0.05)。SCC组术后6h、12h、7d时IL-1βmRNA表达量较Sham组显著性上调(P<0.05),SNCG mRNA表达量较Sham组显著性下调(P<0.05)。术后7d,IL-1βSH组SNCG mRNA表达水平显著高于Vector组(P<0.05)。IL-1βSH组损伤脊髓前角SNCG免疫阳性细胞数和IOD值在术后3d、7d、28d均优于Vector组,差异具有统计学意义(P<0.05)。GeneMANIA分析发现IL-1β和SNCG通过Cfd存在亚细胞定位关系。结论:IL-1βsi RNA可改善SCC大鼠后肢运动功能,上调SNCG蛋白和mRNA的表达,这可能与调节神经元可塑性、抑制线粒体凋亡途径有关。

Objectives: To observe the effect of interleukin-1β(IL-1β) small interfering RNA(siRNA) on motor function in rats after spinal cord contusion(SCC), and to investigate the corresponding mechanism. Methods: A total of 98 female SD rats was randomly divided into four groups: Sham group, SCC group, Vector group and IL-1β SH group. Rats in IL-1β SH group and Vector group were locally injected with IL-1β siRNA or vector in spinal cord at 48 hours before surgery. The vertebra resection was only performed in Sham group and the T10 contusion was prepared in other three groups. Basso Beattie & Bresnahan (BBB) score was recorded for five rats in each group at the day of surgery and 1d, 3d, 5d, 7d and 14d after surgery. In Sham group, rats were sacrificed at 28d after surgery, and the relative expression levels of IL-1β and synuclein gamma(SNCG) mRNA were detected by real-time fluorescence quantitative polymerase chain reaction( qRT-PCR). In SCC group, the relative expression levels of IL-1β and SNCG mRNA were detected at 6h, 12h, 1d, 3d, 5d, 7d and 14d after SCC. In Vector group and IL-1β SH group, the relative expression level of SNCG mRNA was detected at 7d after surgery, and the contused spinal cord was also perfused and fixed at 3d, 7d and 28d after surgery. Immunohistochemistry (IHC) was used to observe the SNCG immune- positive cells. The cells were then counted, and the average integrated optical density(IOD) values were determined. GeneMANIA analyzed the theoretical relationship between IL-1β and SNCG. Results: The BBB score in Sham group was always 21 at each time point. After SCC, the score of SCC group were significantly lower than that of Sham group (P<0.05). The score of IL-1β SH group was significantly higher than that of Vector group at 3d, 5d, 7d and 14d after SCC(P<0.05). The expression of IL-1β mRNA in SCC group was significantly more up-regulated than that in Sham group at 6h, 12h and 7d after surgery respectively(P<0.05), while the expression of SNCG mRNA at each time point was significantly more down-regulated than that in Sham group (P<0.05). The level of SNCG mRNA in IL-1β SH group was significantly higher than that in Vector group at 7d after surgery(P<0.05). In IL-1β SH group, SNCG immunoreactive cells and IOD values in the anterior horn of injured spinal cord were significantly better than those in Vector group at 3d, 7d and 28d after surgery (P<0.05). The subcellular localization between IL-1β and SNCG through Cfd was predicted by GeneMANIA. Conclusions: IL-1β siRNA improves the hindlimb motor function in rats, and upregulated the expression of SNCG protein and mRNA, which may be related to regulation of neuronal plasticity and inhibition of mitochondrial apoptosis pathways.