局灶性脑缺血再灌注损伤后存活素与生长相关蛋白43表达抑制神经细胞凋亡的实验

Expressions of survivin and growth associated protein-43 to inhibit the apoptosis of neurons after cerebral ischemia/reperfusion injury in rats

目的:观察局灶性脑缺血再灌注损伤后脑神经组织存活素及生长相关蛋白43的表达情况及其与细胞凋亡和轴突再生的关系。方法:实验于2006-03/10在青岛大学医学院脑血管病研究所完成。①实验分组:将80只成年健康雄性Wistar大鼠,分为存活素组和生长相关蛋白43组,每组40只。再用随机数字表法分为正常对照组、假手术组和缺血1h再灌注2,6,12,24,48h,3,7,14d组,每组4只大鼠。②实验干预:缺血再灌注各组应用线栓法制备大脑中动脉缺血再灌注模型,假手术组除不插尼龙线外,余步骤同缺血再灌注组。假手术组于术后1d取脑组织,缺血再灌注组大鼠于再灌注2,6,12,24,48h,3,7,14d取脑组织检测相关指标。③实验评估:采用免疫组织化学方法检测存活素与生长相关蛋白43在神经元凋亡中的表达情况。结果:80只大鼠均进入结果分析。①细胞凋亡:缺血再灌注2～24h组海马、皮质区及纹状体区凋亡细胞均增多,再灌注48h,3d组凋亡细胞呈显著增加,再灌注7d组凋亡细胞达高峰,再灌注14d组凋亡细胞显著降低。②存活素表达:Survivin主要分布在细胞质,呈棕黄色或棕褐色。在缺血梗死区灶周围局部较大区域内阳性细胞呈弥漫性分布,缺血中心区较多可见。再灌注2h组海马、皮质区及纹状体区Survivin表达明显增加,6～24h强阳性细胞表达逐渐增加,48h达高峰,3～7d表达恒定,14d表达降低。③生长相关蛋白43表达:缺血再灌注2h脑顶叶皮质区及纹状体区均有生长相关蛋白43阳性神经元表达增加,主要分布于脑梗死灶的周围区。皮质区的阳性神经元发出微丝诱导细胞沿其生长方向定向性地穿过胼胝体向海马迁徙。海马结构内生长相关蛋白43免疫反应阳性产物的密度较高,在齿状回分子层内带染色较深,增粗的强阳性反应的纤维存在于梗死灶的边缘,并向梗死灶中心区伸延。再灌注7d达高峰,14d达最低值。缺血再灌注48h～7d损伤区域神经元轴突呈出芽征,发出突触纤维。缺血再灌注2h～14d纹状体区均高于海马区及皮质区。结论:存活素和生长相关蛋白43在抑制神经元凋亡动态时相的表达中具有非特异性反应并促进神经元的修复和再生。

ATM： To explore the expressions of survivin and growth associated protein （GAP）-43 to inhibit the apoptosis of neurons and promote regeneration of axon after cerebral ischemia/reperfusion （I/R） injury in rats. METHODS： The experiment was carried out in the Institute of Cerebrovascular Disease; Affiliated Hospital of Qingdao University Medical College from March to October in 2006. （1）Eighty healthy adult male Wistar rats were divided into survivin group and GAP-43 group, each contained 40 rats, and they were randomly assigned into control group, sham-operation group and I/R groups, in which ischemia for 1 hour and reperfusion for 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 3 days, 7 days, 14 days, each set contained 4 rats.（2）The models with cerebral I/R were induced by intraluminal middle cerebral artery occlusion with a nylon monofilament suture. The sham-operation group was identical with I/R group except the nylon suture. The brain tissues in sham-operation group were sampled one day postoperation, those of I/R group were performed after the reperfusion was completed. （3）lmmunohistochemistry technique was used to detect the expressions of survivin and GAP-43. RESULTS： Eighty rats were all involved in the result analysis.（1）Cell apoptosis of neurons in hippocampus, cortical and striatal areas was all increased during 2-24 hours of reperfusion, significantly increased during 48 hours to 3 days, peaked at 7 days, and significantly decreased at 14 days.（2）Survivin expression： Survivin mainly distributed in cytoplasm and colored as buffy or brown. The expression of survivin positive neurons was diffused on a large area surrounding ischemia infarction lesion and mostly distributed in ischemia central region. The expression of survivin positive neurons in hippocampus, cortical and striatal areas increased at 2 hours of reperfusion, reached a peak at 48 hours, constant during 3-7 days, and decreased to the lowest level at 14 days.（3）GAP-43 expression： GAP-43 positive neurons in cortical area of parietal lobe and striatal area were increased at 2 hours of reperfusion, mainly distributing surrounding the cerebral infarction lesion. With the microfilament, the neurons in the cortical area induced the cells migrate to hippocampus directionally through corpus callosum. The GAP-43 positive for immunological reaction was dense in hippocampus, and deeply stained in molecular layer of dentate gyrus. The thickening fibers, which were strongly positive, localized at the edge of infarction lesion and extended to the central lesion. The expression peak appeared at 7 days of reperfusion and declined to the lowest at 14 days. The lesion area appeared the budding sign of neurons axon during 48 hours to 7 days of I/R, and they grew synaptic fiber. During 2 hours to 14 days of I/R, the expressions of striatal area was higher than that of hippocampus and cortical area. CONCLUSION： The expressions of survivin and GAP-43 at dynamic state phase can inhibit apoptosis of neurons in a nonspecific reaction and promote the repair and regeneration of neurons.