泛素特异性蛋白酶25在颞叶癫痫大鼠颞叶皮质中表达变化的实验研究

Experimental study on the expression of ubiquitin-specific protease-25 in temporal cortex of epileptic rats

目的探讨海人酸点燃杏仁核大鼠颞叶癫痫模型颞叶皮质中泛素特异性蛋白酶25(USP25)的表达情况。方法将52只SD雄性大鼠按随机数字表法分为癫痫模型组(共39只)和假手术对照组(简称对照组,13只)。癫痫模型组建立海人酸点燃杏仁核大鼠颞叶癫痫模型,再按构建成功的时间分为3组(构建成功1 d为急性期组、构建成功7 d为潜伏期组、构建成功30 d为慢性期组,每组各13只),在观察结束时取材。对照组在杏仁核注入生理盐水,与癫痫模型组伴随取材。免疫组织化学染色及免疫荧光双标法检测USP25在颞叶皮质中的表达及与神经元(NeuN)和星形胶质细胞(GFAP)的共定位情况;实时荧光定量PCR法和蛋白免疫印迹法检测USP25在颞叶皮质中的表达变化。结果在注药侧颞叶皮质中,癫痫模型组USP25与NeuN的共定位阳性细胞在癫痫后期增加,USP25 mRNA及蛋白水平在癫痫发作不同时期的表达差异均有统计学意义(F值分别为25.48、7.68,均P<0.05)。与对照组(mRNA:1.00±0.36;蛋白:1.00±0.46)比较,潜伏期组(mRNA:10.80±4.82;蛋白:1.88±0.32)和慢性期组(mRNA:12.97±4.48;蛋白:1.92±0.26)的表达水平显著增加,差异均有统计学意义(均P<0.05)。在未注药侧皮质中,USP25 mRNA及蛋白水平在癫痫发作不同时期的表达差异也均有统计学意义(F值分别为86.86、6.65,均P<0.05)。结论颞叶皮质中USP25的表达在癫痫大鼠潜伏期后增加,提示去泛素化通路参与颞叶癫痫的慢性病理过程。

Objective To study the expression of ubiquitin-specific protease 25(USP25)in the temporal cortex of the kainic acid(KA)induced epilepsy rat model.Methods Fifty-two male SD rats were randomly divided into the epilepsy model group(n=39)and sham-operated control group(n=13)with the random number table.The epilepsy model group was established by injecting KA into the amygdala,then the epileptic rats were randomly divided into 3 groups according to the modeling success time:1 day for acute period,7 days for latent period and 30 days for chronic period(13 rats in each group).Those rats were sampled at the end of observation.Rats in the control group were injected with normal saline into the amygdala and sampled together with those in the experimental group.Immunohistochemistry and immunofluorescence double labeling was used to test USP25 expression and its co-expression with neurons(NeuN)and astrocytes(GFAP).Quantitative real-time PCR and Western blot were used to assess the change of USP25 in the temporal cortex of rats.Results In the ipsilateral temporal cortex,the positive cells of co-expression of USP25 and NeuN were increased in the later stage of epilepsy in the epilepsy model group,and the expression levels of USP25 mRNA and protein in different stages of epilepsy varied significantly(F=25.48 and 7.68 respectively,both P<0.05).Compared with the control group(mRNA level:1.00±0.36,protein level:1.00±0.46),the expression of USP25 in the latent group(mRNA level:10.80±4.82,protein level:1.88±0.32)and the chronic group(mRNA level:12.97±4.48,protein level:1.92±0.26)were increased significantly(all P<0.05).In the contralateral temporal cortex,the expression levels of USP25 mRNA and protein in different stages of epilepsy also varied significantly(F=86.86 and 6.65 respectively,both P<0.05).Conclusions The increased expression of USP25 in the temporal cortex after the latent period has suggested that the deubiquitination pathway is involved in the chronic pathological process of temporal lobe epilepsy.