P-糖蛋白在大鼠脑缺血再灌注后的表达变化

目的 观察大鼠局灶性脑缺血90 min再灌注后p-糖蛋白(P-gp)在脑组织中的表达及其变化规律.方法 采用线栓法建立大鼠脑缺血再灌注模型,选取26只成年健康SD雄性大鼠,其中1只用于2,3,5--三苯基氯化四氮唑(TTC)染色,其余25只随机分为对照组(n=5)、假手术组(n=5)、脑缺血再灌注1、3、7d组(n=5).用免疫组织化学DAB单标,观察P-gp在正常脑组织和缺血脑组织中的分布变化;用Mdr-1抗体分别和神经元标记物(Neun)、星形胶质细胞标记物(GFAP)、微血管内皮细胞标记物(CD31)进行免疫荧光双标,观察P-gp在缺血再灌注后脑组织的表达;用实时定量PCR技术检测脑缺血再灌注后大脑皮质及纹状体微血管P-gp mRNA的表达变化.结果 对照组仅在脑组织微血管内皮细胞表达P-gp,缺血再灌注组除微血管内皮细胞表达P-gp外,在部分神经元及星形胶质细胞的突起也有表达.脑缺血再灌注后1d皮质P-gp mRNA表达降低,但3d表达明显增高,7d又下降,其升高和降低水平与对照组及假手术组比较,差异有统计学意义(P＜0.05).脑缺血再灌注后1、3、7d纹状体P-gp mRNA表达均增多,其中以1、3d增多明显,与对照组和假手术组比较,差异有统计学意义(P＜0.05).结论 脑缺血再灌注后P-gp在大脑皮层及纹状体微血管P-gp的表达呈现不同的趋势,这可能是脑组织的自我保护机制之一.

Changes of expression of P-glycoprotein after cerebral ischemia reperfusion in rats

Objective To observe the changes of the expression of P-glycoprotein(P-gp) after 90 min focal cerebral ischemia reperfusion in rats.Methods The model of.focal cerebral ischemia induced by blocking middle cerebral artery was made by using an intraluminal filament technique.A total of 26 adult SD male rats was used for experiment.One of them was applied for the 2,3,5-triphenyl tetrazolium chloride(TTC) staining to detect whether the focal cerebral ischemia model was successfully made,and the remaining rats were randomly divided into control group(n =5),sham operation group(n =5),and cerebral ischemia reperfusion for 1,3,7 d group(n=5).The immunohistochemistry single staining was used to observe the changes of P-gp in the normal and repefued ischemial brain tissue.The immunofluorescence double staining was used to observe the expression of P-gp in the normal and repefued ischemial brain tissue with Mdr-1 antibody,Neun antibody(marker of the neuron),GFAP antibody(marker of the astrocytes),and CD31 antibody(marker of capillary endothelium).Meanwhile the changes of P-gp in ischemic cerebral cortex and striatum capillary were analyzed by using real-time quantitative PCR technique.Results In control group,P-gp was only located in cerebral microvascular endothelial cells,while in cerebral ischemia reperfusion group,it also could be detected in some neurons and astrocytes.After cerebral ischemia reperfusion,the mRNA expression of P-gp in cerebral cortex was decreased on day 1,significantly increased on day 3,and then decreased on day 7.There was significantly statistical difference of the mRNA expression of P-gp cortex in cerebral ischemia reperfusion for 1,3,7 d group compared with control group and sham group(P＜0.05).After cerebral ischemia reperfusion,the mRNA expression of P-gp in cerebral striatum was increased on day 1,day 3and day 7,especially on day 1 and day 7,and the difference in cerebral ischemia reperfusion group was statistically significant compared with the other two groups (P＜0.05).Conclusion P-gp can only be expressed in cerebral microvascular endothelial cells in normal rats,while it can also be expressed in neurons and astrocytes in rats after the brain's subjection to ischemia reperfusion,and it showed different tendencies between cortex and striatum,which may be regarded as one of the self-protection mechanisms in brain tissue.