鞘氨醇激酶1在颞叶癫痫中的作用机制研究

Probing the mechanism of sphingosine kinase 1 in temporal lobe epilepsy

目的观察鞘氨醇激酶1(SphK1)在难治性颞叶癫痫(TLE)患者及匹罗卡品诱导的TLE大鼠模型中的表达,探讨其在TLE发病中的作用机制。方法收集TLE患者手术切除的皮质标本,纳入癫痫组(n=16)。收集脑外伤患者切除的颞叶皮质标本,纳入对照组(n=10)。将72只雄性SD大鼠按随机数字表法分为模型对照组(MC组,n=32)和匹罗卡品组(PILO组,n=40),PILO组根据匹罗卡品诱导癫痫持续状态(SE)后的观察时间随机分为4个亚组:E6h组、E1d组、E3d组和E7d组(n=8)。采用免疫组化染色法检测SphK1在TLE患者颞叶皮质中的表达变化;运用Western blotting法检测SphK1在大鼠海马中的表达变化;采用免疫荧光染色法观察在人颞叶皮质和大鼠海马中星形胶质细胞(AST)活化增生情况和SphK1在AST中的表达。结果癫痫组人颞叶皮质中SphK1的阳性细胞数及AST细胞数均明显多于对照组(均P<0.05)。E6h组、E1d组、E3d组和E7d组大鼠海马SphK1表达水平均明显高于MC组(均P<0.05);PILO组AST细胞数明显多于MC组(P<0.05)。免疫荧光染色结果显示,在癫痫组患者颞叶皮质中,SphK1主要在活化的AST的胞质中表达;而在PILO组大鼠海马中,SphK1主要在活化的AST的胞核中表达。结论 SphK1在TLE患者颞叶皮质和TLE大鼠海马中的表达明显增加,SphK1参与了TLE的发病。

Objective To observe the expression of sphingosine kinase 1(SphK1) in brain tissue of temporal lobe epilepsy(TLE) patients and pilocarpine-induced TLE rats, and to investigate the pathogenesis of TLE. Methods Temporal lobe cortex samples from 16 patients with TLE were collected as epilepsy group and temporal lobe cortex samples from 10 patients with brain trauma were used as control group. Seventy-two male rats were randomly divided into model control group(MC group, n=32) and Li-Pilocarpine group(PILO group, n=40). The MC group and PILO group were divided into 4 subgroups: E6 h group, E1 d group, E3 d group and E7 d group(n=8). Immunohistochemistry was used to detect the expression of SphK1 in human temporal lobe cortex. Western blotting technique was used to measure the expression of SphK1 in rat hippocampus at different point of time after pilocarpine treatment. Immunofluorescence was applied to detect the activation and proliferation of astrocytes(AST) and the localization of SphK1 in human temporal lobe cortex and rat hippocampus. Results The numbers of SphK1 positive cells and AST cells in human temporal cortex in epilepsy group were significantly higher than those in control group(all P<0.05). SphK1 expressions of rats hippocampus in E6 h group, E1 d group, E3 d group and E7 d group were significantly higher than that in MC group(all P<0.05). The number of AST cells in rats hippocampus in PILO group was significantly higher than that in MC group(P<0.05). The immunofluorescence staining showed that in the human temporal lobe cortex of epilepsy group, SphK1 was mainly expressed in the cytoplasm of activated AST;In the hippocampus of rats in the PILO group, SphK1 was mainly expressed in the nucleus of activated AST. Conclusions SphK1 is overexpressed in temporal lobe cortex of TLE patients and TLE rat hippocampus. SphK1 may play an important role in the pathogenesis of temporal lobe epilepsy.