干细胞来源外泌体通过调控MMP-9表达减轻脊髓损伤的机制分析  

Mechanism analysis of stem cell-derived exosomes attenuating spinal cord injury by regulating MMP9 expression

作　　者：王东 郜振武 杨秋荟 孙留群 杨学斌 陈晨 WANG Dong;GAO Zhen-wu;YANG Qiu-hui(Department of Spine Surgery,Quwo County Traditional Chinese Medicine Hospital,Linfen 043400,China)

机构地区：[1]曲沃县中医医院脊柱外科,043400 [2]白求恩医院骨科,030001 [3]临汾市中心医院骨科,041099

出　　处：《中国现代药物应用》2022年第21期179-182,共4页Chinese Journal of Modern Drug Application

基　　金：山西白求恩医院科研项目(项目编号:2021YJ18)。

摘　　要：目的探讨干细胞来源外泌体通过调控基质金属蛋白酶-9(MMP-9)表达减轻脊髓损伤的机制。方法60只SD大鼠,按随机数字表法分为假手术组、模型组及外泌体组,每组20只。以改良Allen's法建立大鼠脊髓损伤模型,假手术组不对脊髓进行打击,仅行T10椎板切除术;外泌体组在脊髓损伤30 min和1 d后进行干细胞来源外泌体尾静脉注射;模型组分别于损伤后30 min、1 d经尾静脉注射磷酸缓冲盐溶液(PBS)。于损伤后第3天采用伊文思蓝染色观察血脊髓屏障通透性;通过Western blot检测MMP-9、紧密连接蛋白(claudin-5、Occludin、ZO-1)的表达水平,明胶酶谱法检测MMP-9活性;采用Basso-Beattie-Bresnahan运动等级(BBB)评分量表评估大鼠在损伤后第1、3、5、7、10、14、21天的运动功能。①比较三组大鼠脊髓损伤后不同时间点的BBB评分、染料渗出量、紧密连接蛋白(claudin-5、ZO-1、Occludin)表达水平及脊髓组织中MMP-9表达水平、活性。结果假手术组大鼠无行动障碍,BBB评分均为21分。外泌体组、模型组大鼠脊髓损伤后运动功能均受损,第1天BBB评分为0分,两组大鼠脊髓损伤后第3、5、7天BBB评分比较差异无统计学意义(P<0.05);外泌体组大鼠脊髓损伤后第10、14、21天BBB评分高于模型组,差异有统计学意义(P<0.05)。三组大鼠损伤后第3天血脊髓屏障染料渗出量及claudin-5、ZO-1、Occludin相对表达量比较差异具有统计学意义(P<0.05)。外泌体组大鼠脊髓损伤后第3天血脊髓屏障染料渗出量(38.11±2.10)μg/g少于模型组的(58.40±6.15)μg/g,差异有统计学意义(P<0.05);外泌体组大鼠claudin-5、ZO-1及Occludin表达水平分别为(1.17±0.28)、(1.11±0.12)、(1.26±0.33),高于模型组的(0.49±0.14)、(0.70±0.19)、(0.78±0.22),差异有统计学意义(P<0.05)。外泌体组大鼠脊髓组织中MMP-9表达水平(1.20±0.11)低于模型组的(1.72±0.15),差异有统计学意义(P<0.05);外泌体组大鼠脊髓组织Objective To discuss the mechanism of stem cell-derived exosomes attenuating spinal cord injury by regulating matrix metalloproteinase-9(MMP-9)expression.Methods A total of 60 SD rats were divided into sham operation group,model group and exosome group according to the random numerical table,with 20 rats in each group.A rat model of spinal cord injury was established by the modified Allen's method.The sham operation group was not subjected to a blow to the spinal cord,and only underwent T10 laminectomy;the exosome group underwent tail vein injection of stem cell-derived exosomes at 30 min and 1 d after spinal cord injury;the model group was injected with phosphate buffered saline(PBS)via tail vein at 30 min and 1 d after injury.On the 3rd day after injury,Evans blue staining was used to observe the permeability of the blood spinal cord barrier,Western blot was used to detect the expressions of MMP-9 and tight junction proteins(claudin-5,Occludin,ZO-1),gelatin zymography was used to detect the activity of MMP-9,and the Basso-Beattie-Bresnahan(BBB)locomotor rating scale was used to evaluate the motor function recovery of rats on the 1st,3rd,5th,7th,10th,14th and 21st days after injury.Comparison was made on BBB score,amount of dye exudation,expression levels of tight junction proteins(claudin-5,ZO-1,and Occludin)at different time points after spinal cord injury,as well as the expression level and activity of MMP-9 in spinal cord tissue among the three groups.Results The rats in the sham operation group had no mobility impairment,and the BBB score was 21 points.The motor function of the rats in the exosome group and the model group was impaired after spinal cord injury,and the BBB score was 0 on the first day.There was no significant difference in the BBB score between the two groups on the 3rd,5th,and 7th days after spinal cord injury(P<0.05).On the 10th,14th,and 21st days after spinal cord injury,the BBB scores of rats in the exosome group were higher than those in the model group,and the differences were statisti

关 键 词：干细胞 外泌体 基质金属蛋白酶-9 脊髓损伤 血脊髓屏障 

分 类 号：R651.2[医药卫生—外科学]