發(fā)布時(shí)間：2018-09-07 09:12

【摘要】：目的探討microRNA-34b及caspase-9在TPM(托吡酯)治療癲癇大鼠海馬組織中表達(dá)的差異及其作用的研究。方法將100只健康成熟的雄性SD大鼠隨機(jī)分成四組:正常組(n=25),癲癇組(n=25),TPM低劑量組(n=25)及TPM高劑量組(n=25)。采用鋰-匹羅卡品誘發(fā)大鼠癲癇發(fā)作,選取癲癇發(fā)作IV-V級,并持續(xù)抽搐30min的大鼠為實(shí)驗(yàn)組。將致癲成功的大鼠平均分成3組,對TPM高劑量組(80mg/kg.d)、低劑量組(40mg/kg.d)每日同一時(shí)間灌胃制作藥物組模型,正常組及癲癇組予生理鹽水(4ml/kg.d)灌胃,灌胃28d后取大腦及海馬組織。HE染色觀察大鼠海馬組織的病理改變;采用芯片分析法、實(shí)時(shí)定量PCR法觀察各組海馬組織中microRNA-34b的差異性表達(dá);利用蛋白免疫印跡法及免疫組化染色檢測各組海馬中caspase-9的變化。結(jié)果1.芯片分析結(jié)果發(fā)現(xiàn)：與正常對照組相比,癲癇組中部分microRNA(34b、204、2964、207、210、351、764、511)是上調(diào)的,部分 microRNA(136、219、382、337、31、383、499、30b、582、708、488、598)是下調(diào)的。不同劑量TPM治療后,上調(diào)的microRNA部分出現(xiàn)了下調(diào),下調(diào)的microRNA部分出現(xiàn)了上調(diào),其中microRNA-34b表達(dá)較為明顯。2.實(shí)時(shí)定量PCR檢測結(jié)果;與正常對照組相比,癲癇組中microRNA-34b的表達(dá)顯著上調(diào),差異有明顯的統(tǒng)計(jì)學(xué)意義(p0.01)。經(jīng)高劑量TPM治療后,microRNA-34b的表達(dá)與癲癇組相比明顯下調(diào)(p0.01),低劑量TPM治療后,microRNA-34b的表達(dá)與癲癇組相比也發(fā)生了明顯下調(diào)(p0.01),其中,高劑量TPM組比低劑量TPM組microRNA-34b下調(diào)的明顯(p0.05)。3.蛋白免疫印跡法:癲癇組大鼠海馬組織中caspase-9蛋白表達(dá)明顯高于正常組(p0.01)。與癲癇組對比,低劑量TPM組中caspase-9蛋白的表達(dá)是下降的(p0.05),而高劑量TPM組中caspase-9蛋白的表達(dá)與癲癇組相比顯著下降(p0.01),其中,高劑量TPM組比低劑量TPM組caspase-9蛋白表達(dá)下降的更為明顯(p0.01)。4.HE染色:正常對照組可見大小均一、形態(tài)規(guī)整、排列整齊的細(xì)胞。相反,癲癇組大鼠齒狀回DG區(qū)顆粒細(xì)胞,海馬回CA1區(qū)小椎體細(xì)胞、CA3區(qū)大椎體細(xì)胞排列疏松紊亂、細(xì)胞腫脹、邊緣不清,胞核、胞質(zhì)深染,染色質(zhì)聚集成塊,細(xì)胞成空泡樣改變。而經(jīng)TPM治療后,細(xì)胞形態(tài)有所改善,其中,高劑量TPM組比低劑量TPM組細(xì)胞形態(tài)改善的更為明顯。5.免疫組化:癲癇組中caspase-9蛋白的表達(dá)明顯高于正常對照組(p0.01)。低劑量TPM組中,caspase-9蛋白的表達(dá)與癲癇組相比是下降的(p0.05),高劑量TPM組中,caspase-9蛋白的表達(dá)與癲癇組相比下降的更為顯著(p0.01),其中,高劑量TPM組比低劑量TPM組caspase-9蛋白表達(dá)下降的明顯(p0.05)。結(jié)論1.癲癇大鼠海馬中,microRNA-34b出現(xiàn)了上調(diào)。經(jīng)TPM治療后,microRNA-34b出現(xiàn)了下調(diào),且隨TPM劑量的增加,microRNA-34b表達(dá)的更明顯。2.TPM預(yù)處理后caspase-9表達(dá)升高,提示其神經(jīng)保護(hù)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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R742.1

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