【作者】 张睿；

【导师】 黄永辉；

【作者基本信息】 江苏大学 ， 外科学（专业学位）， 2020， 硕士

【摘要】 目的:验证缺血后适应(Ischemic post-conditioning,IPC)对大鼠脊髓缺血再灌注损伤(Spinal cord ischemia reperfusion injury,SCIRI)的保护作用并检测其对钙敏感受体(Calcium-sensing receptor,CaSR)及内质网应激相关标志物Caspase-12表达的影响。方法:五十只SD大鼠被完全随机化等分为5个组(为充分避免因不同动物个体之间的差异而产生随机误差):假手术组(Sham组),缺血再灌注模型组(IR组),IR模型+缺血后适应组(IPC组),IPC组+激动剂(IPC-GdCl3组)和IR模型+抑制剂组(NPS2390组)。Sham组在手术中只需显露大鼠腹主动脉的一段而不用动脉夹阻断血流,其余各组阻断腹主动脉25分钟左右后再松开,构建脊髓IR模型;IPC组于恢复血流3-4分钟后给予三个周期的缺血后适应操作;IPC-GdCl3组在连续2天尾静脉注射CaSR激动剂GdCl3后行IPC组操作;对于NPS2390组我们采取的干预方法是提前2天,每天通过尾静脉注射一定量的NPS2390,随后建立IR模型。通过3B评分法对大鼠后肢运动功能进行评价来推测脊髓再灌注损伤的程度区别,我们选用TUNEL法检测脊髓组织中细胞凋亡数目并通过蛋白质免疫印迹技术及免疫组织化学定量检测CaSR及Caspase-12表达后记录数据予以分析。结果:1.除Sham组以外其余4组大鼠3B评分均少于Sham组(P<0.05),对于IPC组及NPS2390组,分数则分别多于IR组和IPC-GdCl3组(P<0.05)。2.TUNEL法阅片记录显示,假手术组仅能看到零星的阳性反应;IR组和IPC-GdCl3组阳性细胞数目多于IPC组及NPS2390组(P<0.05)。3.蛋白免疫印迹检测结果可见,建模各组CaSR及Caspase-12蛋白印迹表达量显著高于假手术组(P<0.05);IR组及IPC-GdCl3组的CaSR及Caspase-12表达量分别高于IPC组及NPS2390组(P<0.05)。4.免疫组织化学结果可见,除Sham组以外的其余建模各组CaSR及Caspase-12蛋白表达高于假手术组(P<0.05);IR组及IPC-GdCl3组的CaSR及Caspase-12表达量分别高于IPC组及NPS2390组(P<0.05)。结论:1.缺血后适应能够对大鼠脊髓缺血再灌注损伤起到一定程度的恢复作用并改善相关功能损害;2.缺血后适应在保护过程中可抑制脊髓组织中CaSR及Caspase-12的表达。更多还原

【Abstract】 Objective:To verificate the protective effect of spinal cord ischemic postconditioning（IPC）on spinal cord ischemia reperfusion injury（SCIRI）in rats and to observe its effect on the expression of calcium-sensing receptor（CaSR）and endoplasmic reticulum stress-related proteins Caspase-12.Methods:Fifty SD rats were completely randomized into 5 groups（to fully avoid random errors due to differences between different animal individuals）:sham operation group（Sham group）,ischemia reperfusion model group（IR Group）,IR model+post-ischemic adaptation group（IPC group）,IPC group+agonist（IPC-GdCl₃group）and model+inhibitor group（NPS2390 group）.Sham group only exposed abdominal aorta but not clamping,SCIRI models were established after clamping abdominal aortic 30min and then openning it in other groups,IPC group was treated with 3 circulatory IPC after 3min perfusion,IPC-GdCl₃ group was treated with IPC group’s treatment after tail vein injection of CaSR agonist GdCl₃ for 2 days.CaSR inhibitor NPS2390 was injected intravenously in NPS2390 group for 2 days before establishing model.The motor function of hindlimb was evaluated by BBB score,the apoptosis of spinal cord tissue was detected by TUNEL staining,and the expression of CaSR and Caspase-12 was detected by Western blot and immunohistochemistry.Results:1.The BBB scores of the other 4 groups were lower than those of the Sham group（P<0.05）.The scores for IPC and NPS2390 groups were higher than those for IR and IPC-GdCl3 groups respectively（P<0.05）.2.TUNEL results showed that few apoptotic cells were found in the Sham group,and the number of apoptotic cells in the IR and IPC-GdCl3 groups was higher than that in the IPC and NPS2390 groups（P<0.05）.3.Western blot results showed that the expression levels of CaSR and Caspase-12in each group were apparently higher than those in the sham operation group（P<0.05）;The expression of CaSR and Caspase-12 in IR group and IPC-GdCl₃ group was higher than that in group IPC and group NPS2390（P<0.05）.4.The results of immunohistochemistry showed that the expression of CaSR and Caspase-12 proteins in modeling groups was higher than that in sham operation groups（P<0.05）;CaSR and Caspase-12 expression in IR and IPC-GdCl3 groups were higher than those in IPC and NPS2390 groups（P<0.05）.Conclusions:1.Ischemic postconditioning can relieve spinal cord ischemia reperfusion injury in rats.2.Ischemic postconditioning inhibits CaSR and Caspase-12 expression in spinal cord tissue during protection.更多还原