热激活蛋白70识别蛋白在脊髓缺血再灌注损伤后表达变化及其意义

Expression of heat shock cognate protein 70 after spinal cord ischemia-reperfusion injury

中文关键词:  脊髓缺血再灌注损伤  热激活蛋白70识别蛋白  应激

英文关键词:Spinal cord ischemia-reperfusion injury  Heat shock cognate protein 70  Stress

中文摘要:

  【摘要】 目的：探讨热激活蛋白70识别蛋白（heat shock cognate protein 70，HSC70）在兔脊髓缺血再灌注损伤（spinal cord ischemia-reperfusion injury，SCIRI）后表达变化及其意义。方法：随机将36只新西兰白兔分成6组（每组6只），采用Zivin法建立兔SCIRI模型。A组只显露腹主动脉而不阻断血流，30min后关闭腹腔，取材（L3～L5段脊髓）；B组阻断腹主动脉血流30min后关闭腹腔，取材；缺血再灌注组（C、D、E、F组）阻断腹主动脉血流30min后停止夹闭，再灌注6、12、24、48h时采用改良Tarlov评分分别进行运动功能评价后取材。应用差异蛋白质组学技术荧光差异双向凝胶电泳联合质谱分析筛选出应激相关蛋白HSC70，利用免疫印迹对质谱进行印证，结合免疫组化研究其在脊髓内的时空变化特点。结果：成功建立SCIRI兔模型，SCIRI后实验动物后肢功能逐渐好转，C～F组改良Tarlov评分分别为1.167±0.753、1.667±0.516、2.668±0.516和2.167±0.752分，再灌注24h达SCIRI早期最好水平，再灌注48h略有下降。应激相关蛋白HSC70在A组印迹清晰，B组印迹轻微加强，C组和D组印迹再次加强，E组印迹显著减弱至最低，F组印迹灰度回归至再灌注6~12h水平。A组灰质中部体积较小中间神经元胞浆见轻度免疫反应；B组灰质各部位神经元胞浆免疫反应程度与A组相当；C组胶质细胞核免疫反应加重；D组神经元免疫反应减轻，但胶质细胞及雪旺细胞核免疫反应较强烈；E组神经元免疫反应进一步减弱，低于A组水平；F组神经元胞浆免疫反应加重，胶质细胞及雪旺细胞核免疫反应仍强烈。结论：HSC70参与SCIRI发生和发展过程，可作为SCIRI预防、诊断和治疗的靶点。

英文摘要:

  【Abstract】 Objectives: To investigate the significance and variation of heat shock cognate protein 70(HSC70) expression after spinal cord ischemia-reperfusion injury. Methods: Thirty-six New Zealand white rabbits were randomly divided into six groups, and established SCIRI models by using the Zivin′s method. In group A, the abdominal aorta was revealed without blood blocked, marrow was obtained after abdominal closure. In group B, blood in abdominal aorta was blocked for 30 minutes before abdominal closure, then marrow was obtained. In the ischemia-reperfusion groups(C, D, E, F), blood was blocked for 30 minutes before reperfusion, motor function was evaluated by modification Tarlov score at 6h, 12h, 24h, 48h after reperfusion, then marrow was taken in each group. Adopted two-dimensional fluorescence difference gel electrophoresis to obtain the HSC70. Immunohistochemistry and Western blot were employed to analyze the role of HSC70 in SCIRI and provide theoretical evidence for the prevention, diagnosis and treatment of SCIRI. Results: SCIRI rabbits were set up successfully. The modified Tarlov scores at group C, D, E, F was 1.167±0.753, 1.667±0.516, 2.668±0.516 and 2.167±0.752 respectively. The function of experimental animals′ hind legs was gradually improved after SCIRI and reached the early best level after 24 hours reperfusion, while declined slightly after 48 hours reperfusion. The immunblot gray scale of HSC70 was clear in group A, strengthened slightly in group B, strengthened again in group C and D. But gray scale of group E decreased to the lowest, and that of group F returned to the level of 6~12 hours reperfusion. Small interneurons cytoplasm of central gray matter in group A had mild immune response; the cytoplasm of neurons of grey matter in group B had the same immune responses as group A; immune response of glial cells nucleus in group C was aggravated; immune response of neurons in group D decreased, but that of glial cells and Schwann cells nucleus was strong; immune response of neurons in group E was further weakened, and below the level of group A; immune response of neuronal cytoplasm in group F was aggravated, while that of glial cells and Schwann cells nucleus was still strong. Conclusions: HSC70 involves in the occurrence and development of SCIRI and it can be the target of prevention, diagnosis and treatment of SCIRI.