建立兔脊髓缺血再灌注损伤模型PDIA3的表达变化及临床意义  

Expression and clinical significance of protein disulfide-isomerase A3 in a rabbit model of spinal cord ischemia/reperfusion injury

作　　者：李春旭[1] 张善勇[1] 齐治平[1] 夏鹏[1] 潘肃[1] 昝春芳 杨小玉[1] 

机构地区：[1]吉林大学第二医院骨科,吉林省长春市130041

出　　处：《中国组织工程研究》2017年第24期3845-3850,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(31572217;81350013);吉林省科技型中小企业技术创新基金(SC201502001);吉林大学研究生创新基金资助项目(2017176)~~

摘　　要：背景:目前认为脊髓缺血再灌注损伤是造成脊髓减压手术后"二次瘫痪"的主要原因,而控制应激相关蛋白和兴奋性氨基酸对脊髓缺血再灌注损伤的治疗至关重要。目的:观察蛋白质二硫键异构酶A3(PDIA3)在兔脊髓缺血再灌注损伤后表达变化。方法:依据Zivin法建立兔脊髓缺血再灌注损伤模型,将36只新西兰白兔随机均分成6组,对照组只显露腹主动脉而不阻断血流,30 min后关闭腹腔;实验组阻断腹主动脉血流30 min后分别再灌注0,6,12,24,48 h再关闭腹腔。首先均利用改良Tarlov评分进行运动功能评价;随后取损伤段腰髓(L3-L5),结合荧光差异双向凝胶电泳和质谱分析筛选出PDIA3;其次应用免疫印迹印证质谱;最后通过免疫组织化学结果观察其在脊髓内的时间和空间表达变化特点。结果与结论:(1)运动功能评分结果:实验动物于脊髓缺血再灌注损伤发生后均可见后肢功能逐渐好转现象,评分结果为缺血再灌注24 h达最高水平,48 h又略有下降;(2)免疫印迹结果:对照组PDIA3表达有清晰印迹显示,缺血再灌注0 h印迹轻度增强,6-12 h进一步增强,24 h显著减弱至最低水平,48 h再次升高到6-12 h水平;(3)免疫组织化学结果:神经元胞浆中可见PDIA3表达,且中间神经元表达量明显多于运动神经元;(4)结果表明:脊髓缺血再灌注损伤发生发展过程中PDIA3异常上调,说明PDIA3与脊髓缺血再灌注损伤密切相关,可作为其诊断和治疗的新靶点。BACKGROUND： At present, spinal cord ischemia/reperfusion injury is considered as the main reason for ＂secondary paralysis＂ after spinal decompression, and to control the levels of stress-related proteins and excitatory amino acids plays an important role in the treatment of spinal cord ischemia/reperfusion injury. OBJECTIVE： To investigate the expression level of protein disulfide-isomerase A3 （PDIA3） after spinal cord ischemia/reperfusion injury in rabbits. METHODS： Thirty-six New Zealand white rabbits were enrolled, the models of spinal cord ischemia/reperfusion injury were established using Zivin＇s method, and were then randomized into six groups （n=6 per group）. The rabbit abdominal aorta in control group was exposed without vascular occlusion and then the abdominal cavity was closed 30 minutes later. In experimental groups, the abdominal aorta was blocked for 30 minutes, followed by 0, 6, 12, 24 and 48 hours of reperfusion, and then the abdominal cavity was closed. The neurological function was evaluated with a modified Tarlov score. The L^s lumbar vertebrae were removed, and PDIA3 was screened by two-dimensional fluorescence differential gel electrophoresis combined with mass spectrometry, and then its temporal and spatial changes in the spinal cord were detected by western blot assay and immunohistochemistry. RESULTS AND CONCLUSION＂ The function of hind limbs was improved in all the experimental groups after spinal cord ischemia/reperfusion injury, and the modified Tarlov scores reached the peak at 24 hours after schemia/reperfusion injury, and decreased slightly at 48 hours. The expression of PDIA3 in the control group showed clear imprinting, which was slightly strengthened at 0 hour, became more strengthened at 6-12 hours, significantly reduced to the minimum level at 24 hours, and returned to the level of 6-12 hours at 48 hours after ischemia/reperfusion. Immunohistochemical results showed that there was visible PDIA3 in the cytoplasm of neurons, and the expression level in the in

关 键 词：脊髓 缺血 再灌注损伤 蛋白质二硫键异构酶 组织构建 组织工程 脊髓缺血再灌注损伤 蛋白质二硫键异构酶A3 应激 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]