【作者】 凡进；

【导师】 殷国勇；

【作者基本信息】 南京医科大学 ， 骨科学， 2010， 硕士

【摘要】 目的:研究BAD蛋白(Bcl - xL/ Bcl- 2 associated death promoter, BAD)在脊髓缺血再灌注损伤中的作用机制,并进一步探讨是否可以通过抑制JNK活性来抑制BAD蛋白的活性从而保护神经细胞。方法:新西兰白兔随机分为三组:假手术组(5只)、缺血再灌注组(缺血前2h给予溶于PBS的25% DMSO硬膜外注射,按再灌注不同时间段分为4组,每组5只,分别为:A组0.5h、B组2h、C组8h、D组24h),JNK抑制组(缺血再灌注2h前注射SP600125(溶于PBS的25% DMSO,1.0 mg/kg),分组同缺血再灌注组)。光镜观察各组脊髓标本的形态学改变,电镜观察各组脊髓标本及线粒体的形态学改变,Western blot检测各组脊髓标本中JNK及p- JNK、BAD及p-BAD、cyto C、Bcl-xL、Bcl- 2、14-3-3的表达,免疫共沉淀检测各组脊髓标本中BAD与14-3-3、BAD与Bcl-xL/Bcl- 2之间的结合与分离,免疫组化观察p-BAD及14-3-3的定位表达。结果:ⅰ)脊髓标本的电镜结果显示:脊髓组织分别在缺血再灌注组B组、JNK抑制组D组中开始出现组织浸润,间质间出血等现象。ⅱ)脊髓标本的电镜结果显示:脊髓神经细胞分别在缺血再灌注组B组、JNK抑制组D组中开始出现细胞核浓缩、染色质聚边,脱髓鞘改变等早期凋亡征像。ⅲ)线粒体的电镜结果显示:线粒体分别在缺血再灌注组B组、JNK抑制组D组中开始出现固缩及向核周聚集等凋亡征像。ⅳ)Western blot结果显示:p-JNK、cyto C在缺血再灌注组B组开始表达且表达量随缺血时间的延长而增高,在JNK抑制组中则在D组开始表达且活性小于缺血再灌注组。p-BAD的去磷酸化在缺血再灌注组B组开始激活且表达量随缺血时间的延长而降低,在JNK抑制组中则在D组开始激活且活性小于缺血再灌注组。ⅴ)免疫共沉淀结果显示:BAD分别在缺血再灌注组B组、JNK抑制组D组中开始与14-3-3逐渐分离,同时逐渐与Bcl-xL、Bcl- 2结合。ⅵ)免疫组化结果显示:BAD和14-3-3在假手术组的胞浆中大量表达,在缺血再灌注组中从B组开始表达下降,JNK抑制组则在D组才可见表达降低。结论:(1)脊髓缺血再灌注损伤时,BAD蛋白被激活,与14-3-3的结合分离而与Bcl-xL、Bcl-2形成二聚体,促使细胞色素C从线粒体释放至胞浆内,诱发了以线粒体为中心的细胞凋亡程序。(2)JNK抑制剂在抑制JNK活性的同时可抑制BAD蛋白的活性,进而减少缺血再灌注损伤过程中脊髓神经细胞的凋亡。更多还原

【Abstract】 Objective:To elucidate the role and mechanism of BAD in reperfusion of ischemic spinal cord, and investigate the the neuroprotective effect of JNK inhibitor by inhibition of the activity of BAD.Methods:Forty-five white adult New England rabbits were randomly assigned to one of the three groups: Group I: sham-operation group(n = 5), Group II: Ischemic reperfusion group: 25% DMSO in PBS was intrathecally injected 2 h prior to the ischemic injury, (ischemia 30 minutes/reperfusion 30 minutes group( group A, n=5) , ischemia 30 minutes/reperfusion 2 hour group( group B, n=5) and ischemia 30 minutes/reperfusion 8 hours group ( group C, n=5), ischemia 30 minutes/reperfusion 24 hours group ( group D, n=5)),and Group III :JNK inhibitor group:SP600125(1.0 mg/kg) in 25% DMSO in PBS was intrathecally injected in the JNK inhibitor group 2 h prior to the ischemic injury,Specific groups are exactly the same as Group II. Changes in spinal cord morphology were observed by Hematoxylinand eosin (HE) stain and Electron Microscopy; mitochondrial morphology were observed by Electron Microscopy; the activation of p- JNK, JNK and BAD, p-BAD、cytochrome C、Bcl-xL、Bcl-2、14-3-3 was detected by Westernblot; the interactions between 14-3-3 and BAD, BAD and Bcl-XL or Bcl-2 were performed by coimmunoprecipitation analysis; The localization of 14-3-3 and p-BAD were analyzed by immunohistochemistry.Results:ⅰ)For HE stain, hemorrhagic focuses were found and the neuronal cells were swollen in group B, C, D of ischemic reperfusion group and in group D of JNK inhibitor group.ⅱ) Electron microscopic examination of spinal cord revealed demyelination, coarse chromatin condensation, and breakdown of the nucleus into discrete fragments in group B, C, D of ischemic reperfusion group and in group D of JNK inhibitor group.ⅲ) Electron microscopic examination of mitochondria shows that the cristae appeared dense, and the boundaries of mitochondria were irregular in group B, C, D of Ischemic reperfusion group and in group D of JNK inhibitor group.ⅳ) In ischemic reperfusion group,the expressions of p-JNK and cytochrome C were increased, and enhanced With the extension of time was gradually increasing in group B, C, D of ischemic reperfusion group and in group D of JNK inhibitor group. While the expression of p-BAD was decreased.ⅴ) The dissociation of ASK1 from 14-3-3 and the Bcl-XL/BAD and Bcl-2/BAD dimerization were increased, and subsequently increased in group B, C, D of ischemic reperfusion group. While the decrease was observed in group D of JNK inhibitor group.ⅵ) p-BAD and 14-3-3 were mainly observed in neuronal cells in the sham-operation group. The amount of cytoplasmic staining of p-BAD and 14-3-3 was decreased in group B of ischemic reperfusion group, and subsequently decreased in group C and group D of ischemic reperfusion group. While the decrease was observed in group D of JNK inhibitor group. Conclusion:(1) The activity of BAD is induced in the reperfusion of ischemic spinal cord, which lead to the decrease in BAD/14-3-3 dimerization, the increase in dimerization of BAD with Bcl-XL and Bcl-2, the release of cytochrome c, and the neurocyte apoptosis via the mitochondrion-dependent mechanism.(2) JNK inhibitor could inhibit the apoptosis of neurocytes by inhibition of the activity of BAD.更多还原