【作者】 刘秉锐；

【导师】 马玉林；

【作者基本信息】 宁夏医科大学 ， 外科学， 2009， 硕士

【摘要】 目的通过建立大鼠脊髓缺血再灌注损伤模型,检测再灌注损伤区脊髓组织不同时间点转录核因子(NF-κB),细胞间粘附分子-1( ICAM-1)和血管细胞间粘附分子-1(VCAM-1)蛋白表达的变化,以探讨其表达时间规律及调控机制。同时应用中药甘草酸二铵进行干预治疗,探讨其是否有神经保护作用及其机制。为临床治疗脊髓损伤提供实验研究数据和新的治疗思路。材料和方法在实验1中,40只健康雄性(体重220-250g) SD大鼠随机分为正常组(5只)、假手术组(5只)和缺血再灌注组(30只)。缺血再灌注组又按再灌注0h, 3h, 24h,48h,72h和168h分为六个亚组,每亚组5只。正常组不做任何处理;假手术组为只打开腹腔显露腹主动脉不阻断即关腹,3小时后取材;缺血再灌注组通过外科手术途径用无创动脉夹阻断左右肾动脉之间的腹主动脉,30min后解除,从而造成大鼠腰段脊髓缺血再灌注损伤。各组取腰段脊髓标本,用免疫组织化学方法观察NF-κB, ICAM-1, VCAM-1蛋白的表达,并进行相关性分析。同时进行组织病理学及电镜检查,观察脊髓前角神经元的病理和超微机构的变化。72h和168h亚组取标本之前应用改进Behrmann 5点评分法评价大鼠后肢功能。在实验2中,模型制作同实验1。40只健康雄性SD大鼠(体重220-250g)随机分为缺血再灌注(IR)组(20只)和甘草酸二铵治疗(DG)组(20只);各组又按再灌注3h,24h, 72h,168h分为四亚组,每亚组5只。DG组大鼠于缺血前10分钟由舌下静脉按20mg/kg注射甘草酸二铵,IR组大鼠于缺血前10分钟由舌下静脉注射等量生理盐水。各组取腰段脊髓标本,用免疫组织化学方法观察NF-κB, ICAM-1, VCAM-1蛋白的表达,光镜观察组织学变化。72h和168h亚组取标本之前应用改进Behrmann 5点评分法评价大鼠后肢功能。分析比较甘草酸二铵是否有神经保护作用。结果(1)脊髓缺血再灌注后各时间点脊髓组织样本均有明显病理改变,可见组织水肿,神经元空泡形成及坏死神经元(2)脊髓缺血再灌注后各时间点脊髓组织样本NF-κB及ICAM-1, VCAM-1表达均增加,NF-κB于再灌注后24h表达达高峰,ICAM-1于再灌注后48h表达达高峰,VCAM-1于再灌注后48-72h表达达高峰,随后降低,但再灌注后168h三者表达仍高于正常水平。(3) NF-κB的表达与血管内皮ICAM-1, VCAM-1的表达呈正相关(r=0.800;r=0.496 , P < 0.01)。(4)甘草酸二铵能显著降低脊髓缺血再灌注损伤后NF-κB , ICAM-1及VCAM-1的表达。对再灌注3、24、72和168 h脊髓组织中NF -κB p65表达的抑制率分别是15.40%、20.17%、19.28%和11.11%。(4)甘草酸二铵能明显促进大鼠脊髓缺血再灌注损伤后后肢功能的恢复,对大鼠后肢功能平均恢复率分别为27.59%和19.35%。结论(1)脊髓缺血再灌注损伤后NF-κB, ICAM-1, VCAM-1大量表达,是脊髓缺血再灌注损伤重要机制之一,NF-κB的活化能提高血管内皮细胞ICAM-1, VCAM-1蛋白表达。(2)甘草酸二铵可能通过降低NF-κB, ICAM-1和VCAM-1的表达来发挥其神经保护作用。更多还原

【Abstract】 Objective By establishing the rat’s models of spinal cord ischemia-reperfusion injury(SCII),We investigate the rule and modulating mechanism of the expressions of NF-κB、ICAM-1 and VCAM-1 through detecting their expressions after different time of ischemia-reperfusion. We also explore the neuroprotective effects of Diammonium Glycyrrhizinate (DG). Then dicuss whether DG has protective effect on SCII and provide experimental data and new idea for clinical prevention and cure of spinal cord injury.Materials and methods In experiment one, forty healthy SD male rats, weighing 220-250 g, were randomly divided into normal group(n=5),sham operation(SO) group(n=5) and ischemia-reperfusion (IR)group(n=30), then IR group was divided into six(0h, 3h, 24h,48h,72h and 68h) subsets(n=6)according to time after ischemic-reperfusion injury. The normal group were not any controlled;the SO group were opended peritoneal cavity but not intercepted abdominal aorta;the IR group were intercepted abdominal aorta between right and left renal arter with bulldog clamp for 30min. The lumbar myeloid tissues were prepared in the different groups,respectively. The expressions of NF-κB p65、ICAM-1 and VCAM-1 in lumbar myeloid tissues were analyzed by immunohistochemistry . Then analyze the correlation between NF-κB p65 and ICAM-1, VCAM-1.Meanwhile the histopathological chances in spinal cord were observed by hematoxylin and eosin (HE) staining. Microstructure changes of neuron was observed by electron microscope.In experiment two, a model of spinal cord ischemic-reperfusion injury was completed with intercepting rat’s abdominal aorta between right and left renal arter for 30 min. Fourty healthy SD male rats, weighing 220-250 g, were randomly divided into IR group and DG group(n=20).Each rat was injected 20 mg/kg DG via subl ingual vein 10 minutes before ischemia occurred in DG group, equal qualities of physiological saline in the IR group.The two groups were observed at 3, 24, 72 and 168h after ischemia-reperfusion, respectively. Lumbar myeloid tissues were prepared at the different times, respectively. The expression of NF-κB p65、ICAM-1 and VCAM-1 in lumbar myeloid tissues were analyzed by immunohistochemistry and histopathological changes of spinal cord were observed by HE staining. Meanwhile, each rat’s hind limb function was evaluated at 72 and 168 hours after ischemia-reperfusion with Behrmann’s methods in two groups.Result (1) HE staining showed obvious histological changes of lumbar myeloid tissues of each subset after ischemic-reperfusion injury . myeloid tissue edema, vacuolus and neuron necrosis were observed .(2)The expressions of NF-κB p65、ICAM-1 and VCAM-1 in lumbar myeloid tissues strengthened after ischemic-reperfusion injury. After ischemic-reperfusion NF-κBp65 immunoreactivity reached the peak at 24h, ICAM-1 at 48h, VCAM-1 at 48-72h, respectively. then all weakened slowly, but remained a higher level than normal level. (3) The expression of NF-κB in myeloid tissues was positively correlated with the expression of ICAM-1 and VCAM-1 in vascular endothelial cell (r=0.80;r=0.496, P < 0.01).(4) DG has depression effect for the expression of NF-κB、ICAM-1 and VCAM-1 in myeloid tissues after ischemia-reperfusion. The inhabitation ratio of NF-κB p65 expression by DG was 15.40%, 20.17%, 19.28% and 11.11% at 3, 24, 72 and 168 hours after ischemia-reperfusion, respectively.(5) DG has promotive effect on the functional recover of hind limb .The promotion ratio of hind limb function by DG was 27.59% and 19.35% at 72 and 168 hours after ischemia-reperfusion,respectively.Conclusion (1) High expressions of NF-κB p65、ICAM-1 and VCAM-1 may play a key role in the spinal cord ischemia-reperfusion injury. The activation of NF-κB p65 may be involved in the modulating mechanism of the expressions of ICAM-1 and VCAM-1 in vascular endothelial cell. (2) DG may reduce the spinal cord damage after ischemia-reperfusion and has neuroprotective effects on the spinal cord. It may play its neuroprotective role by reducing the expressions of NF-κB p65、ICAM-1 and VCAM-1.更多还原