摘要
目的探讨IL-17参与脊髓损伤后免疫炎症反应的作用机制,在细胞因子水平为临床治疗脊髓损伤提供新的靶点。方法雄性C57BL/6小鼠随机分为4组:模型组制作小鼠脊髓钳夹模型,假手术组只剪开硬脊膜不伤及脊髓,IL-17中和抗体组在脊髓钳夹1 h即刻尾静脉给予IL-17中和抗体,溶剂对照组在脊髓钳夹1 h尾静脉给予0.01μmol/L无菌PBS。小鼠后肢的行为学评分(mouse scale for locomotion,BMS)观察各组小鼠1~7 d后肢行为学变化,免疫组织化学技术观察各组小鼠脊髓损伤第7天脊髓Neu N神经元的形态学变化,实时荧光定量PCR检测脊髓损伤区第7天IL-1β、IL-6、TNF-αm RNA的表达变化。结果小鼠脊髓损伤后,行为学检测结果显示:假手术组小鼠BMS评分1~7 d均为9分,模型组、IL-17中和抗体组、溶剂对照组小鼠脊髓损伤第1天BMS评分为0分,随着时间的延长,各组小鼠的后肢运动功能均有改善,且IL-17中和抗体组小鼠后肢运动功能改善情况呈优于假手术组以外其他各组趋势。免疫组织化学染色显示:脊髓损伤后第7天,假手术组脊髓灰质内可见大量结构完整的Neu N染色阳性细胞;模型组和溶剂对照组小鼠脊髓灰质中,Neu N染色阳性细胞明显皱缩、突起消失,大量Neu N神经元呈细胞空泡状,数量大幅度减少;IL-17中和抗体组可见部分神经元细胞形态正常,具有完整的Neu N神经元胞体和分支的突触,且Neu N神经元染色阳性细胞数量回升。脊髓损伤后第7天实时荧光定量PCR显示:与假手术组相比,模型组、PBS溶剂对照组IL-1βm RNA显著升高(P〈0.01);与模型组、PBS溶剂组相比,IL-17中和抗体组IL-1βm RNA明显下降(P〈0.05);与假手术组相比,模型组TNF-αm RNA显著升高(P〈0.01);与假手术组相比,PBS溶剂对照组TNF-αm RNA明显升高(P〈0.05);与模型组相比,IL-17中和抗体组TNF-αm RNA表达明显下降(P〈0.05);IL-17中和抗体组IL-6 m RNA水平呈下降趋势,但各组IL-6 m RNA的表达未见统计学差异。结论 IL-17和IL-1β、IL-6、TNF-α共同作用加重了脊髓损伤的免疫炎症进程,抑制IL-17可减轻小鼠脊髓损伤。
Objective To investigate the role of IL-17 in immune inflammatory reaction after spinal cord injury,and provide more evidence for clinical treatment of spinal cord injury on cytokine levels. Methods Male C57BL/6 mice were randomly divided into 4 groups: in the spinal cord injury group,mice were made into spinal cord clamp model. In the sham surgery group,the dura was cut without injuring the spinal cord. The IL-17 neutralizing antibody group received IL-17 neutralizing antibody injection through the cadual vein at 1 h after the spinal cord clamp. The solvent control group received the sterile PBS(0.01 μmol/L) through the cadual vein at 1 h after the spinal cord clamp. Mouse scale for locomotion(BMS)was applied to evaluate the mice's behavior change of hindlimb in 1-7 d. The real time fluorescent quantitative PCR was used to detect the expression changes of IL-1β、IL-6 and TNF-αm RNA,and the immunohistochemistry technique was conducted to observe the morphological changes of neurons of Neu N on the 7th day after spinal cord injury respectivly. Results The behavior score of mice after spinal cord injury indicates: the BMS scores were all 9 on the 1st to the 7th day in the sham surgery group,but were 0 on the 1st day in the model group,the IL-17 neutralizing antibody group and the solvent control group. With time extension,the motor function of hindlimbs of mice in each group were improved,but improved even better in the IL-17 neutralizing antibody group than in the model group and the solvent control group.Immunohistochemistry staining showed that after spinal cord injury,there were much complete structure of Neu N positive staining cells in the gray matter in the sham surgery group,which were obviously shrinking and protrusions disappearing in the model group and the solvent control group,while large number of Neu N neurons vacuolated and reduced significantly. It could be seen that part of neurons morphology was normal and with complete Neu N neuronal cell bodies and branches of the synapse,and the amount of Neu N neuron staining positive cells rebounded in the IL-17 neutralizing antibody group. The results of RT-q PCR on the 7th day after spinal cord injury indicated that compared with sham surgery group,IL-1β m RNA increased significantly in the model group and the solvent control group(P〈0.01);compared with the model group and the solvent control group,IL-1β m RMA decreased significantly in the IL-17 neutralizing antibody group(P〈0.05);compared with sham surgery group,TNF-α m RNA increased significantly in the model group(P〈0.01);compared with the sham surgery group,TNF-α m RNA increased significantly in the solvent control group(P〈0.05);compared with the model group,TNF-α m RNA decreased significantly in the IL-17 neutralizing antibody group(P〈0.05). IL-6 m RNA expression was on the decline in the IL-17 neutralizing antibody group,but without statistically significant difference with other groups. Conclusion Combined action of IL-17,IL-1β,IL-6 and TNF-α deteriorates the immune inflammatory of spinal cord injury,and it might relieve spinal cord injury in mice by inhibition of IL-17.
出处
《国际药学研究杂志》
CAS
CSCD
北大核心
2015年第2期183-188,共6页
Journal of International Pharmaceutical Research
基金
国家自然科学基金青年科学基金项目(81101420)