NBP对器官型脑片糖氧剥夺模型中细胞凋亡的影响

The effection of NBP on apoptosis of organotypic brain slices in the model of oxygen-glucose deprivation

目的观察正丁基苯酞（NBP）对乳大鼠大脑皮质器官型脑片的氧糖剥夺模型（OGD）中细胞凋亡和线粒体电位的影响。方法建立SD乳鼠大脑皮质运动区的器官型脑片,SMI-32染色观察锥体细胞。2 w后,随机分为对照组（C组）和实验组（T组）,T组在OGD干预30 min后给予不同剂量NBP,分为T0、T0.01、T0.1、T1、T10组,给NBP前和后1 h观察PI染色;选择T0组、T10组,观察两组在OGD 30 min、NBP 1 h、24 h、72 h、7 d等不同时间点PI染色变化。于NBP后1 h各T组进行JC-1线粒体膜电位检测。结果器官型脑片OGD 30 min给予NBP 1 h后,C组和各T组PI染色可见不同亮度红色荧光,各T组荧光强度均大于C组,荧光强度与NBP浓度（0~10μmol/L范围内）呈负相关,各T之间组及与C组相比差异有显著性（P〈0.05）;T0组和T10组在OGD 30 min均出现明显细胞凋亡,两组无显著性差异。复氧复糖后随时间延长T0组细胞凋亡增加,T10组逐渐减少,差异显著（P〈0.05）;C组和各T组JC-1线粒体检测试剂盒染色可见OGD 30 min,复氧复糖1 h后,红色荧光/绿色荧光（R/G）的值明显减小,R/G的值与NBP浓度呈正相关,各T组R/G值均小于C组,差异显著（P〈0.05）。结论 OGD模型可模拟缺血性脑损伤的病理变化,重复性良好;NBP对缺血性脑损伤导致的急性和迟发性细胞凋亡均具有保护作用;NBP对脑片损伤后线粒体电位的耗散有阻止作用,且与剂量相关。

Objective To establish the oxygen and glucose deprivation（ OGD） model of organotypic brain slices of rat cerebral cortex and observe the effects of n-butyl phthalide（ NBP） on apoptosis and mitochondrial membrane potential.Methods Culturing brain slices of SD rats on the membrane insert and observed the morphological changes of pyramidal cells by SMI-32 stain with immunohistochemistry. After being cultured two weeks in vitro,brain slices were divided into two groups： control group（ group C） and test group（ group T）. Group T include five subgroup（ T0、T0. 01、T0. 1、T1、T10） according to different concentrations of NBP（ 0,0. 01μm,0. 1 μm,1 μm,10 μm） to brain slices after treating with OGD 30 min.Brain slices were observed by PI dyeing before and after being given NBP 1 h with different concentrations. Then observing the brain slices in T0 and T10dyeing by PI at different time（ OGD 30 min,NBP 1 h,24 h,72 h,7 d）. Group C and each subgroups of T,treated by OGD and NBP in order（ no PI）,were detected the mitochondrial membrane potential by JC-1kits. Results Brain slices were treating with NBP 1 h after OGD 30 min,they showed different brightness of red fluorescence in control group and test group（ T0、T0. 01、T0. 1、T1、T10） by PI staining. Compared with control group,the fluorescence intensity of each experimental subgroup were greater and negatively related to the concentration of NBP（ P 0. 05）.Observing the apoptosis in slices of T0 and T10at different time（ OGD 30 min,NBP 1 h、24 h、72 h、7 d）,we found the number of cell death had no significant different at OGD 30 mim. After giving normal glucose and oxygen,the brain slices had increasing fluorescence intensity in T0 and decreasing fluorescence intensity in T10 with prolonged time（ P 0. 05）.After OGD 30 min and returned to normal oxygen and glucose 1 h,the slices were detected by mitochondrial membrane potential detection kit（ JC-1 kit）. The value of red / green fluorescence of each group（ C,T0、T0. 01、T0. 1、T1、T10） dramatic reduced correlated positively with dose of NBP. The value of each experimental group was smaller than the control group（ P 0. 05）. Conclusion Treated with OGD,brain slices showed ischemic damage including acute cell death and delayed apoptosis. The model can be established easily. NBP had protective effect on ischemic brain. There is a dose-effect relationship between NBP and brain damage. There was dissipation of mitochondrial membrane potential in brain slices after OGD. NBP could prevent the decline of mitochondrial potential in dose-effect dependent way.