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目的观察肢体远程缺血后处理(LRIP)对局灶性脑缺血再灌注损伤(I/R)大鼠皮质梗死区周围热休克蛋白70(HSP70)的表达定位及阳性细胞表达变化,探讨LRIP发挥脑保护作用的可能分子机制。方法健康成年SD大鼠,随机分为假手术组(sham)、I/R组及LRIP组。实验采用线栓法建立局灶性大脑中动脉脑缺血(1h)再灌注模型,大鼠脑缺血再灌注即刻行双下肢股动脉橡皮筋结扎10min,放松10min,重复3次建立LRIP组模型。于再灌注1d及3d分别断头取脑,Zea longa评分作为判断MCAO模型成功的标准,Garcia神经行为学评分方法检测大鼠神经损伤程度,2,3,5-氯化三苯基四氮唑(TTC)法检测脑梗死体积,Western blotting检测HSP70蛋白表达含量,免疫组织化学和免疫荧光技术用于检测皮质梗死区周围HSP70阳性表达细胞的数目、部位以及类型。结果应用LRIP后,LRIP组与I/R组比较,神经行为学评分明显增加(P<0.05),脑梗死体积显著降低(P<0.05),HSP70蛋白表达明显增加,其中1d组各组差异无统计学意义(P>0.05),3d各组差异有显著统计学意义(P<0.01);HSP70阳性表达主要在梗死区周围神经元、部分血管内皮细胞和星形胶质细胞突起。结论 LRIP可明显改善脑缺血后神经行为学功能,降低脑梗死体积,此作用可能与LRIP上调皮质梗死区周围神经元、血管内皮细胞和星形胶质细胞HSP70表达有关。
Objective To observe the expression and location of heat shock protein 70 (HSP70) and the expression of positive cells around limbal infarct in focal cerebral ischemia-reperfusion (I / R) rats after limb remote ischemic postconditioning (LRIP) Possible molecular mechanism for exerting brain protective effect. Methods Healthy adult SD rats were randomly divided into sham group, I / R group and LRIP group. The focal middle cerebral artery occlusion (1h) reperfusion model was established by the method of thread embolism. Rats in both groups were ligated with rubber band for 10min, then were relaxed for 10min. The model of LRIP group was established . At 1 d and 3 d after reperfusion, the brain was decapitated and the Zea longa score was used as a criterion for judging the success of MCAO model. The severity of nerve injury was detected by Garcia neurological behavioral score method. 2,3,5-triphenyltetrazolium chloride (TTC) method was used to detect the volume of cerebral infarction. Western blotting was used to detect the expression of HSP70 protein. Immunohistochemistry and immunofluorescence were used to detect the number, location and type of HSP70 positive cells around cortical infarct. Results Compared with I / R group, the score of neurobehavioral score of LRIP group was significantly increased (P <0.05), and the volume of cerebral infarction was significantly decreased (P <0.05). The expression of HSP70 protein was significantly increased in LRIP group and I / R group. Statistical significance (P> 0.05). There was significant difference between the three groups in each group (P <0.01). The positive expression of HSP70 was mainly in peripheral neurons, part of vascular endothelial cells and astrocytes in the infarct area. Conclusions LRIP can significantly improve the neurobehavioral function and reduce the volume of cerebral infarction after cerebral ischemia, and this effect may be related to LRIP up-regulating HSP70 expression in peripheral neurons, vascular endothelial cells and astrocytes in the infarcted area of cortex.