【摘要】 目的研究人脑局灶性缺血后海马神经元损伤与凋亡促进因子半胱氨酸蛋白酶3(caspase 3)之间的关系。方法选取因脑梗死而死亡的尸检脑标本48例,并按缺血时间(发病至死亡的时间)分为8组,选取因其他疾病死亡(无脑缺血)的尸检脑标本6例为对照组;应用HE染色来观察海马CA1区神经元形态变化;应用caspase 3免疫组化染色及caspase 3mRNA原位杂交来测定人脑缺血后caspase 3的表达情况;用末端脱氧核糖核酸转移酶介导的缺口末端标记(TUNEL)法标记凋亡神经元;用微管相关蛋白2(MAP 2)的脱失程度反映神经元的受损程度。结果海马CA1区,缺血8h可检测到caspase 3免疫组化染色的阳性细胞(8. 05个/高倍视野), 24h达高峰(24. 85个/高倍视野);caspase 3mRNA原位杂交阳性表达始于4h(6. 75个/高倍视野), 16h达高峰(17. 60个/高倍视野);二者均在72h后呈明显下降趋势。缺血24h可检测到TUNEL阳性细胞,持续至72h。MAP 2免疫活性下降早在4h即可检测到,之后持续下降,至72h几乎无阳性表达细胞。72h前,caspase 3mRNA在TUNEL染色下与时间成正相关,相关系数为0. 721 (P<0. 05 );使用MAP 2时与时间成负相关,相关系数为0. 857(P<0. 05)。4 ~16h,受损神经元形态基本正常; 24 ~48h细胞凋亡特征明显; 72h后,几乎所有神经元形态均呈现严重病理变化?更多还原

【Abstract】 Objective To investigate the correlation between the change of ischemic neuronal injury and caspase-3 post-ischemia in human hippocampus.Methods Forty-eight post-mortem specimens from 48 patients, who died of cerebral infarction were observed. According to ischemic time, the 48 specimens were divided into eight groups. Besides, we selected 6 specimens from 6 patients as control, who died of other diseases without cerebral ischemia. Morphological changes were first analyzed by observing hematoxyline/eosin-staining hippocampal sections. The expression of caspase-3 was investigated by using ~in situ hybridization and immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick-end labeling (TUNEL) method was performed to clarify the involvement of caspase-3 in neuron death. The loss of microtubule-associated protein 2 (MAP-2) was applied to judge the damaged area and degree of neuronal injury induced by ischemia.Results In the CA1 sector of hippocampus, caspase-3 immunostaining modestly increased at 8 hours (8.05/high-power field, hpf), dramatically increased at 24 hours (24.85/hpf), and decreased somewhat after 72 hours. Caspase-3 mRNA was detectable at 4 hours (6.75/hpf), reached a maximum at 16 hours (17.60/hpf), and faded at 72 hours. TUNEL-positive cells were detectable at 24 hours (10.76/hpf), markedly increased at 48—72 hours. The loss of MAP-2 was obviously detected at 4 hours, progressed significantly between 24 and 72 hours; MAP-2 immunoreactivity was barely detectable at 72 hours. Before 72 hours, the caspase-3 evolution was in parallel with both the up-regulation of TUNEL and the loss of MAP-2. The positive correlation between caspase-3 mRNA and TUNEL was significant at the 0.05 level (correlation coefficient was 0.721); the negative correlation between caspase-3 mRNA and MAP-2 was significant at the 0.05 level (correlation coefficient is 0.857). At the early stage (before 72 hours), the staining of caspase-3 mRNA and immunohistochemistry was predominantly present in cytoplasm; the staining of TUNEL was predominantly localized in nucleus. At 4—16 hours, most neurons in hippocampal CA1 areas had relatively normal morphology; at 24—48 hours, neurons showed apoptotic morphology; at 72 hours, most cells showed significantly pathological morphology.Conclusions There existed a time-dependent evolution of neuronal damage after hippocampal ischemia in human brain, which was characterized by its close correspondence to caspase-3.更多还原