CAJ | 학술논문

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NeuN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-αmRNA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NeuN染色阳性细胞；模型组和溶剂对照组小鼠脊髓灰质中，NeuN染色阳性细胞明显皱缩、突起消失，大量NeuN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NeuN神经元胞体和分支的突触，且NeuN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-1βmRNA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-1βmRNA明显下降（P＜0.05）；与假手术组相比，模型组TNF-αmRNA显著升高（P＜0.01）；与假手术组相比，PBS溶剂对照组TNF-αmRNA明显升高（P＜0.05）；与模型组相比， IL-17中和抗体组TNF-αmRNA表达明显下降（P＜0.05）；IL-17中和抗体组 IL-6 mRNA水平呈下降趋势，但各组IL-6 mRNA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。目的：探討IL-17參與脊髓損傷後免疫炎癥反應的作用機製，在細胞因子水平為臨床治療脊髓損傷提供新的靶點。方法雄性C57BL/6小鼠隨機分為4組：模型組製作小鼠脊髓鉗夾模型，假手術組隻剪開硬脊膜不傷及脊髓， IL-17中和抗體組在脊髓鉗夾1 h即刻尾靜脈給予IL-17中和抗體，溶劑對照組在脊髓鉗夾1 h尾靜脈給予0.01μmol/L無菌PBS。小鼠後肢的行為學評分（mouse scale for locomotion， BMS）觀察各組小鼠1～7 d後肢行為學變化，免疫組織化學技術觀察各組小鼠脊髓損傷第7天脊髓NeuN神經元的形態學變化，實時熒光定量PCR檢測脊髓損傷區第7天IL-1β、IL-6、TNF-αmRNA的錶達變化。結果小鼠脊髓損傷後，行為學檢測結果顯示：假手術組小鼠BMS評分1～7 d均為9分，模型組、IL-17中和抗體組、溶劑對照組小鼠脊髓損傷第1天BMS評分為0分，隨著時間的延長，各組小鼠的後肢運動功能均有改善，且IL-17中和抗體組小鼠後肢運動功能改善情況呈優于假手術組以外其他各組趨勢。免疫組織化學染色顯示：脊髓損傷後第7天，假手術組脊髓灰質內可見大量結構完整的NeuN染色暘性細胞；模型組和溶劑對照組小鼠脊髓灰質中，NeuN染色暘性細胞明顯皺縮、突起消失，大量NeuN神經元呈細胞空泡狀，數量大幅度減少；IL-17中和抗體組可見部分神經元細胞形態正常，具有完整的NeuN神經元胞體和分支的突觸，且NeuN神經元染色暘性細胞數量迴升。脊髓損傷後第7天實時熒光定量PCR顯示：與假手術組相比，模型組、PBS溶劑對照組IL-1βmRNA顯著升高（P＜0.01）；與模型組、PBS溶劑組相比， IL-17中和抗體組IL-1βmRNA明顯下降（P＜0.05）；與假手術組相比，模型組TNF-αmRNA顯著升高（P＜0.01）；與假手術組相比，PBS溶劑對照組TNF-αmRNA明顯升高（P＜0.05）；與模型組相比， IL-17中和抗體組TNF-αmRNA錶達明顯下降（P＜0.05）；IL-17中和抗體組 IL-6 mRNA水平呈下降趨勢，但各組IL-6 mRNA的錶達未見統計學差異。結論 IL-17和IL-1β、IL-6、TNF-α共同作用加重瞭脊髓損傷的免疫炎癥進程，抑製IL-17可減輕小鼠脊髓損傷。
목적：탐토IL-17삼여척수손상후면역염증반응적작용궤제，재세포인자수평위림상치료척수손상제공신적파점。방법웅성C57BL/6소서수궤분위4조：모형조제작소서척수겸협모형，가수술조지전개경척막불상급척수， IL-17중화항체조재척수겸협1 h즉각미정맥급여IL-17중화항체，용제대조조재척수겸협1 h미정맥급여0.01μmol/L무균PBS。소서후지적행위학평분（mouse scale for locomotion， BMS）관찰각조소서1～7 d후지행위학변화，면역조직화학기술관찰각조소서척수손상제7천척수NeuN신경원적형태학변화，실시형광정량PCR검측척수손상구제7천IL-1β、IL-6、TNF-αmRNA적표체변화。결과소서척수손상후，행위학검측결과현시：가수술조소서BMS평분1～7 d균위9분，모형조、IL-17중화항체조、용제대조조소서척수손상제1천BMS평분위0분，수착시간적연장，각조소서적후지운동공능균유개선，차IL-17중화항체조소서후지운동공능개선정황정우우가수술조이외기타각조추세。면역조직화학염색현시：척수손상후제7천，가수술조척수회질내가견대량결구완정적NeuN염색양성세포；모형조화용제대조조소서척수회질중，NeuN염색양성세포명현추축、돌기소실，대량NeuN신경원정세포공포상，수량대폭도감소；IL-17중화항체조가견부분신경원세포형태정상，구유완정적NeuN신경원포체화분지적돌촉，차NeuN신경원염색양성세포수량회승。척수손상후제7천실시형광정량PCR현시：여가수술조상비，모형조、PBS용제대조조IL-1βmRNA현저승고（P＜0.01）；여모형조、PBS용제조상비， IL-17중화항체조IL-1βmRNA명현하강（P＜0.05）；여가수술조상비，모형조TNF-αmRNA현저승고（P＜0.01）；여가수술조상비，PBS용제대조조TNF-αmRNA명현승고（P＜0.05）；여모형조상비， IL-17중화항체조TNF-αmRNA표체명현하강（P＜0.05）；IL-17중화항체조 IL-6 mRNA수평정하강추세，단각조IL-6 mRNA적표체미견통계학차이。결론 IL-17화IL-1β、IL-6、TNF-α공동작용가중료척수손상적면역염증진정，억제IL-17가감경소서척수손상。
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-αmRNA, and the immunohistochemistry technique was conducted to observe the morphological changes of neurons of NeuN 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 NeuN 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 NeuN neurons vacuolated and reduced significantly. It could be seen that part of neurons morphology was normal and with complete NeuN neuronal cell bodies and branches of the synapse, and the amount of NeuN neuron staining positive cells rebounded in the IL-17 neutralizing antibody group. The results of RT-qPCR on the 7th day after spinal cord injury indicated that compared with sham surgery group, IL-1β mRNA 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 mRMA decreased significantly in the IL-17 neutralizing antibody group(P<0.05); compared with sham surgery group , TNF-α mRNA increased significantly in the model group (P<0.01); compared with the sham surgery group, TNF-α mRNA increased significantly in the solvent control group (P<0.05); compared with the model group , TNF-α mRNA decreased significantly in the IL-17 neutralizing antibody group (P< 0.05). IL-6 mRNA 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.