MiR-34b及caspase-9在托吡酯治疗颞叶癫痫大鼠海马组织中的表达及意义

发布时间：2018-09-07 09:12

【摘要】：目的探讨microRNA-34b及caspase-9在TPM(托吡酯)治疗癫痫大鼠海马组织中表达的差异及其作用的研究。方法将100只健康成熟的雄性SD大鼠随机分成四组:正常组(n=25),癫痫组(n=25),TPM低剂量组(n=25)及TPM高剂量组(n=25)。采用锂-匹罗卡品诱发大鼠癫痫发作,选取癫痫发作IV-V级,并持续抽搐30min的大鼠为实验组。将致癫成功的大鼠平均分成3组,对TPM高剂量组(80mg/kg.d)、低剂量组(40mg/kg.d)每日同一时间灌胃制作药物组模型,正常组及癫痫组予生理盐水(4ml/kg.d)灌胃,灌胃28d后取大脑及海马组织。HE染色观察大鼠海马组织的病理改变;采用芯片分析法、实时定量PCR法观察各组海马组织中microRNA-34b的差异性表达;利用蛋白免疫印迹法及免疫组化染色检测各组海马中caspase-9的变化。结果1.芯片分析结果发现：与正常对照组相比,癫痫组中部分microRNA(34b、204、2964、207、210、351、764、511)是上调的,部分 microRNA(136、219、382、337、31、383、499、30b、582、708、488、598)是下调的。不同剂量TPM治疗后,上调的microRNA部分出现了下调,下调的microRNA部分出现了上调,其中microRNA-34b表达较为明显。2.实时定量PCR检测结果;与正常对照组相比,癫痫组中microRNA-34b的表达显著上调,差异有明显的统计学意义(p0.01)。经高剂量TPM治疗后,microRNA-34b的表达与癫痫组相比明显下调(p0.01),低剂量TPM治疗后,microRNA-34b的表达与癫痫组相比也发生了明显下调(p0.01),其中,高剂量TPM组比低剂量TPM组microRNA-34b下调的明显(p0.05)。3.蛋白免疫印迹法:癫痫组大鼠海马组织中caspase-9蛋白表达明显高于正常组(p0.01)。与癫痫组对比,低剂量TPM组中caspase-9蛋白的表达是下降的(p0.05),而高剂量TPM组中caspase-9蛋白的表达与癫痫组相比显著下降(p0.01),其中,高剂量TPM组比低剂量TPM组caspase-9蛋白表达下降的更为明显(p0.01)。4.HE染色:正常对照组可见大小均一、形态规整、排列整齐的细胞。相反,癫痫组大鼠齿状回DG区颗粒细胞,海马回CA1区小椎体细胞、CA3区大椎体细胞排列疏松紊乱、细胞肿胀、边缘不清,胞核、胞质深染,染色质聚集成块,细胞成空泡样改变。而经TPM治疗后,细胞形态有所改善,其中,高剂量TPM组比低剂量TPM组细胞形态改善的更为明显。5.免疫组化:癫痫组中caspase-9蛋白的表达明显高于正常对照组(p0.01)。低剂量TPM组中,caspase-9蛋白的表达与癫痫组相比是下降的(p0.05),高剂量TPM组中,caspase-9蛋白的表达与癫痫组相比下降的更为显著(p0.01),其中,高剂量TPM组比低剂量TPM组caspase-9蛋白表达下降的明显(p0.05)。结论1.癫痫大鼠海马中,microRNA-34b出现了上调。经TPM治疗后,microRNA-34b出现了下调,且随TPM剂量的增加,microRNA-34b表达的更明显。2.TPM预处理后caspase-9表达升高,提示其神经保护作用的存在
[Abstract]:Objective to investigate the expression of microRNA-34b and caspase-9 in hippocampus of epileptic rats treated with TPM (topiramate). Methods 100 healthy and mature male SD rats were randomly divided into four groups: normal group (n = 25), epilepsy group (n = 25), low dose group (n = 25) and TPM group (n = 25). The epileptic seizures were induced by lithium-pilocarpine in rats. The rats with IV-V grade epileptic seizure and continuous convulsion of 30min were selected as experimental group. The successful epileptic rats were divided into three groups. The rats in TPM high dose group (80mg/kg.d) and low dose group (40mg/kg.d) were given intragastric administration of normal saline (4ml/kg.d) at the same time. The histopathological changes of rat hippocampal tissue were observed by HE staining after 28 days of gastric perfusion, and the differential expression of microRNA-34b in hippocampal tissue of each group was observed by microarray analysis and real-time quantitative PCR method. The changes of caspase-9 in hippocampus were detected by Western blot and immunohistochemical staining. Result 1. The results of microarray analysis showed that part of microRNA (34bT204C29207210351764511) was up-regulated in epileptic group compared with normal control group, and part of microRNA (136-219-382C33-3499B) was down-regulated in epileptic group (582708488598). After treatment with different doses of TPM, the up-regulated microRNA was down-regulated and the down-regulated microRNA was up-regulated, among which the expression of microRNA-34b was more obvious. 2. 2. Compared with the normal control group, the expression of microRNA-34b in epilepsy group was significantly up-regulated, and the difference was statistically significant (p0.01). After high dose TPM treatment, the expression of microRNA-34b was significantly down-regulated compared with epilepsy group (p0.01), and the expression of microRNA-34b in low-dose TPM group was significantly lower than that in epileptic group (p0.01). Among them, the expression of microRNA-34b in high-dose TPM group was significantly lower than that in low-dose TPM group (p0.05). Western blot: the expression of caspase-9 protein in hippocampus of epileptic group was significantly higher than that of normal group (p0.01). Compared with epilepsy group, the expression of caspase-9 protein in low dose TPM group was decreased (p0. 05), while that in high dose TPM group was significantly lower than that in epileptic group (p0. 01). The decrease of caspase-9 protein expression in high dose TPM group was more obvious than that in low dose TPM group (p0.01) .4.HE staining showed that the normal control group had uniform size, regular morphology and well-arranged cells. On the contrary, in the epileptic group, the granulosa cells in the DG region of dentate gyrus and the small vertebrae cells in the CA1 area of the hippocampal gyrus were loosely arranged, the cells were swollen, the edges were unclear, the nucleus and cytoplasm were deep stained, the chromatin was accumulated into a mass, and the cells were vacuolated. After treatment with TPM, the cell morphology was improved, among which, the cell morphology of high dose TPM group was significantly improved than that of low dose TPM group. Immunohistochemistry: the expression of caspase-9 protein in epileptic group was significantly higher than that in normal control group (p 0.01). The expression of caspase-9 in low-dose TPM group was lower than that in epileptic group (p0.05), and the expression of caspase-9 protein in high-dose TPM group was significantly lower than that in epileptic group (p0.01). The expression of caspase-9 protein in high-dose TPM group was significantly lower than that in low-dose TPM group (p0.05). Conclusion 1. MicroRNA-34 b was up-regulated in hippocampus of epileptic rats. After TPM treatment, microRNA-34b was down-regulated, and the expression of microRNA-34b increased with the increase of TPM dose. 2. The expression of caspase-9 increased after pretreatment with TPM, indicating the existence of neuroprotective effect.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1

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