摘要
目的:通过观察全脑缺血再灌注后Sprague-Dawley(SD)大鼠海马CA1区脑红蛋白(Ngb)与Caspase-3的时间变化关系,分析脑红蛋白与Caspase-3表达时间的规律性及其两者的相关性机制,为临床抗缺血损伤提供参考依据。
方法:随机将60只健康SD大鼠雌雄各半随机分为全脑缺血/再灌注(I/R)组,假手术(SO)组。每组按其再灌注的时间又分为3、6、12、24、48小时(h)共5个观察时间点,每个时间点6只大鼠,采用改良的Pulsinelli等四血管阻塞法(椎动脉留置针法)建立大鼠脑I/R损伤的模型。两组于再灌注后腹腔注射生理盐水(0.5ml/kg),并每6h注射一次,在相应的时间点断头取脑。用免疫组化法和原位杂交法检测海马CA1区锥体细胞中Ngb及Caspase-3的表达,测量其平均光密度值(OD),用苏木精-伊红(HE)染色检测存活锥体细胞数,用TUNEL原位末端标记法检测凋亡锥体细胞,并计算凋亡率。
结果:在SO组,Caspase-3蛋白及Caspase-3 mRNA、Ngb蛋白及Ngb mRNA各时间点表达的平均光密度值对比差异无统计学意义(P>0.05),存活神经元细胞数及细胞凋亡率的各时间点比较差异均无统计学意义(P>0.05)。在I/R组,Caspase-3蛋白及Caspase-3 mRNA在缺血再灌注后3小时开始表达,24小时达高峰,48小时后逐渐下降,24小时与3、6、12、48小时比较差异有统计学意义(P<0.05);Ngb蛋白及Ngb mRNA在缺血再灌注后3、6小时弥散表达,3、12、24、48小时与SO组比较差异有统计学意义(P<0.05);存活神经元细胞数及细胞凋亡率的各时间点比较差异均有统计学意义(P<0.05)。SO组各时间点的Caspase-3和Ngb比较差异无统计学意义(P>0.05)。I/R组各时间点的Caspase-3和Ngb比较差异有统计学意义(P<0.05)。Ngb与Caspase-3呈负相关(r=-0.882,P<0.05);Ngb与细胞凋亡率呈负相关(r=-0.984,p<0.01); Caspase-3与细胞凋亡率呈正相关(r=0.994,P<0.01)。
结论:(1)脑缺血再灌注损伤后Caspase-3呈时间递增性表达,Ngb呈递减性表达;
(2) Caspase-3与Ngb呈负相关性,Ngb可能抑制细胞的凋亡。
Objective:To investigate the expression of neuroglobin (Ngb) and Caspase-3 in hippocampus CA1 region of the Sprague-Dawley (SD) rats with global cerebral ischemia/reperfusion(I/R) injury and analyze the expression regularity of Ngb and Caspase-3 in differernt time,meanwhile analyze the relevance of Ngb and Caspase-3 distribution;discuss the effect mechanism of neuroglobin and Caspase-3.provide a reference for clinical.
Methods:60 healthy SD rats were randomly divided into 2 groups:sham operation (SO) group and I/R group.each group was assigned into 5 observing time-points: 3,6,12,24,48 hours (h) according to reperfusion time. The SD rats model of global cerebral ischemica/reperfusion was produced by means of simple Pulsinelli-Brierley's four arteries occlusion method.SO and I/R groups were both intraperitoneally injected with 0.9% Sodium chloride (dose:0.5ml/kg) per six hours after reperfusion.Then SD rats'cerebrums were taken out of their skull at each of observing time-point.The expression activation of Caspase-3 and Ngb protein in pyramidal cells in hippocampus comuammonis (CA)1 region were examined by immunohistochemical method (SABC) and Caspase-3 mRNA and Ngb mRNA by in situ hybridization, measured their average optical density(OD),and hematoxylin and eosin (HE) staining was also performed to detect the number of surviving pyramidal cells,and terminal-deoxynucleotidy transferase medatd dUTP nick end labeling (TUNEL) was used to detect apoptotic pyramidal cells (positive cells), And calculated the apoptotic rate of cells.
Results:SO group,the expression of average optical density (OD) of Caspase-3 protein and Caspase-3 mRNA,Ngb and Ngb mRNA were not statistically significant(P>0.05);the number of surviving neurons and apoptotic rate of cells was no significant changes,there was no statistically significant (P>0.05);In the I/R group, Caspase-3 protein and Caspase-3 mRNA began to express at 3h after reperfusion,at the top at 24h,decreased gradually after 48h,24h has variability conspicuous statistically significant compared with3、6、12、48h (P<0.05);Ngb protein and Ngb mRNA's dispersively expressed at 3h,6h after reperfusion,3、12、24、48h compared with the SO group was statistically significant(p<0.05);the number of surviving neurons and apoptotic rate of cells was significant changes,there was statistically significant (P<0.05).In SO group,Caspase-3 compared with the Ngb was not statistically significant(P>0.05). In the I/R group was statistically significant(P<0.05). Correlated analysis showed that there was negative correlation between the expression levels of Ngb and Caspase-3(r=-0.882,p<0.05),Ngb and apoptotic rate of cells(r =-0.984, P<0.01);positive correlation between Caspase-3 and apoptotic rate of cells (r =0.994, P<0.01).
Conclusion:(1) After cerebral ischemia-reperfusion injury Caspase-3'expression was increased with time,meanwhile Ngb was reduced; (2) Caspase-3 has a negative correlation with the Ngb after global cerebal ischemia/reperfusion injury,neuroglobin may inhibit neuronal apoptosis.
引文
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