大鼠脊髓损伤后线粒体自噬和凋亡相关蛋白的表达变化及其发生机制  

Changes and related mechanism of mitophagy-and apoptosis-related protein expressions after spinal cord injury in rats

作　　者：丁轶群 毛腾飞 李喜功[3] 杨晓慧 张钦 Ding Yiqun;Mao Tengfei;Li Xigong;Yang Xiaohui;Zhang Qin(Graduate School,Changzhi Medical College,Changzhi 046000,China;Department of Spine Surgery,Yuncheng Central Hospital(Yuncheng Hospital Affiliated to Shanxi Medical University),Yuncheng 044000,China;Department of Spine Surgery,First Affiliated Hospital of Zhejiang University School of Medicine,Hangzhou 310000,China)

机构地区：[1]长治医学院研究生院,长治046000 [2]运城市中心医院(山西医科大学附属运城医院)脊柱外科,运城044000 [3]浙江大学医学院附属第一医院脊柱外科,杭州310000

出　　处：《中华创伤杂志》2023年第12期1118-1129,共12页Chinese Journal of Trauma

基　　金：山西省自然科学基金(201901D111472);山西省医学重点科研项目(2021XM56);山西省卫生健康委科研课题(2020168)。

摘　　要：目的探讨大鼠脊髓损伤后线粒体自噬和凋亡相关蛋白的表达变化及其发生机制。方法将96只健康雌性SD大鼠按随机数字表法分为假手术组(48只)和脊髓损伤组(48只),每组分为1、3、7、14、21、28 d共6个时间点,各时间点8只。假手术组将T8~9棘突及椎板切除但不损伤脊髓,脊髓损伤组采用Allen法建立脊髓损伤模型。采用BBB评分评估两组伤后各时间点运动功能;HE染色观察两组伤后各时间点脊髓组织病理学改变;免疫荧光观察两组伤后各时间点电压依赖性阴离子通道蛋白1(VDAC1)分别与微管相关蛋白1轻链3(LC3)Ⅱ、E3泛素连接酶(Parkin)、多泛素结合蛋白(p62)共定位阳性细胞情况;Western blot法检测两组各时间点线粒体LC3Ⅱ/LC3Ⅰ、细胞质Parkin、线粒体Parkin、细胞质p62、线粒体p62、细胞质细胞凋亡蛋白(Bax)、线粒体Bax、细胞质细胞色素C(Cyt C)、线粒体Cyt C的表达情况。结果(1)假手术组伤后各时间点BBB评分均为(21.00±0.00)分,脊髓损伤组伤后1、3、7、14、21、28 d BBB评分分别为(0.94±0.50)分、(1.69±0.70)分、(4.13±0.99)分、(11.81±1.03)分、(15.06±1.12)分、(18.38±0.83)分(P<0.01)。(2)与假手术组比较,脊髓损伤组伤后1、3 d脊髓组织结构明显破坏,可见大量出血灶,炎性细胞浸润,神经元肿胀,空洞形成;伤后7、14 d出血灶较前明显减少,神经元胞体肿胀,炎性细胞浸润,存在大量空洞;伤后21、28 d可见大量细胞参与修复,细胞排列紊乱。(3)与假手术组比较,脊髓损伤组伤后1、3 d VDAC1分别与LC3Ⅱ、Parkin、p62共定位阳性细胞数明显增多,此后共定位阳性细胞数逐渐减少。(4)假手术组和脊髓损伤组伤后1、3、7、14、21、28 d线粒体LC3Ⅱ/LC3Ⅰ表达量分别为0.56±0.05和1.00±0.05、1.19±0.11、0.86±0.05、0.80±0.08、0.66±0.13、0.51±0.11,细胞质Parkin表达量分别为0.80±0.13和0.47±0.08、0.29±0.06、0.57±0.07、0.70±0.05、0.97±0.09、0.88±0.12,Objective To explore the changes of mitophagy-and apoptosis-related protein expressions after spinal cord injury in rats and its related mechanism.Methods Ninety-six healthy female SD rats were divided into sham surgery group(n=48)and spinal cord injury group(n=48)according to the random number table.Each group was divided into six time points of 1,3,7,14,21 and 28 days with 8 rats at each time point.In the sham surgery group,the T8-9 spinous processes and vertebral plates were removed without damage to the spinal cord;in the spinal cord injury group,the spinal cord injury model was established using Allen′s method.At each post-injury time point in two groups,BBB score was used to evaluate the motor function of the rats;HE staining was used to observe the histopathological changes of the spinal cord;immunofluorescence was used to observe the co-localized positive cells of the voltage-dependent anion channel protein 1(VDAC1)with microtubule-associated protein 1 light chain 3(LC3)II,E3 ubiquitin ligase(Parkin),and polyubiquitin-binding protein(p62);Western blotting was used to observe expressions of the mitochondrial LC3 II/LC3 I,cytoplasmic Parkin,mitochondrial Parkin,cytoplasmic p62,mitochondrial p62,cytoplasmic apoptotic proteins(Bax),mitochondrial Bax,cytoplasmic cytochrome C(Cyt C),and mitochondrial Cyt C.Results(1)Compared with the sham surgery group[(21.00±0.00)points at all time points],the BBB scores in the spinal cord injury group were(0.94±0.50)points,(1.69±0.70)points,(4.13±0.99)points,(11.81±1.03)points,(15.06±1.12)points and(18.38±0.83)points at 1,3,7,14,21 and 28 days after injury,respectively(P<0.01).(2)Compared with the sham surgery group,the structure in the spinal cord injury group was significantly disrupted at 1 and 3 days after injury,when a large number of hemorrhagic foci,inflammatory cell infiltration,neuronal swelling,and cavity formation were observed.At 7 and 14 days after injury,the number of hemorrhagic foci was markedly reduced compared with the earlier period,when swollen ne

关 键 词：脊髓损伤 线粒体 自噬 细胞凋亡 

分 类 号：R651.2[医药卫生—外科学]