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目的探讨发育期幼鼠反复惊厥后脑内丛生蛋白的表达及溶酶体酶抑制剂E-64 d对其表达的干预作用。方法日龄21 d的SD大鼠随机分为3组:惊厥组(RS组,n=24)、E-64 d干预组(ERS组,n=24)和对照组(CON组,n=19)。于大鼠21日龄开始,RS组大鼠隔日腹腔注射青霉素(5.1×106U.kg-1.d-1),连续6次诱导惊厥发作;ERS组于每次惊厥前腹腔注射4μg E-64 d,采用同样方法腹腔注射青霉素诱导惊厥发作;CON组大鼠予相应剂量的9 g.L-1盐水腹腔注射。于末次惊厥后21 d(出生后51 d),依据Racine标准从RS组及ERS组各随机选取点燃成功的6只大鼠作为本实验的RS组和ERS组。从CON组随机选取6只作为本实验的CON组。处死18只大鼠,并取其大脑海马及皮质,采用免疫印迹技术(Western blot)检测18只大鼠大脑海马、皮质中丛生蛋白的表达。各组蛋白水平应用SPSS 17.0软件进行统计学分析。结果 RS组大鼠海马及皮质丛生蛋白表达水平均较CON组显著增高,差异均有统计学意义(Pa<0.05)。ERS组海马丛生蛋白的表达低于RS组,差异有统计学意义(P<0.05);ERS组皮质丛生蛋白的表达与RS组比较差异无统计学意义(P>0.05)。结论丛生蛋白参与惊厥性脑损伤的过程,E-64 d的作用可能与其下调丛生蛋白的表达有关。
Objective To investigate the expression of clusterin in brain after recurrent seizures and the effect of lysosomal enzyme inhibitor E-64 d on its expression. Methods Sprague - Dawley (SD) rats were randomly divided into 3 groups: seizure group (RS group, n = 24), E-64 d intervention group (n = 24) and control group ). The rats in RS group were injected intraperitoneally with penicillin (5.1 × 106U.kg-1.d-1) every other day for 6 consecutive times to induce seizures. Rats in RS group were injected intraperitoneally with 4 μg of E-64 d, using the same method of intraperitoneal injection of penicillin convulsion seizures; CON rats to the corresponding dose of 9 gL-1 saline intraperitoneal injection. At 21 d after the last convulsion (51 d after birth), six rats were randomly selected from the RS group and the ERS group according to the Racine standard to serve as the RS group and the ERS group. Six rats were randomly selected from the CON group as the CON group. Eighteen rats were killed and their hippocampus and cortex were taken. The expression of clusterin in hippocampus and cortex of 18 rats was detected by Western blot. Each group of protein levels using SPSS 17.0 software for statistical analysis. Results The expression of hippocampus and cortical clusterin in RS group was significantly higher than that in CON group (P <0.05). The expression of clumsy protein in the ERS group was lower than that in the RS group (P <0.05). There was no significant difference between the RSS group and the ERS group (P> 0.05). CONCLUSIONS: Clusterin is involved in the process of convulsive brain injury. The effect of E-64 d may be related to the down-regulation of the expression of clusterin.