【摘要】：目的:癲癇是由神經(jīng)元網(wǎng)絡(luò)異常放電引發(fā)的神經(jīng)系統(tǒng)疾病,對(duì)個(gè)人、家庭和社會(huì)帶來很大的負(fù)擔(dān),癲癇的有關(guān)發(fā)病機(jī)制至今尚未明確�？p隙連接是細(xì)胞之間聯(lián)系的通道,承載著許多信息交換,研究表明縫隙連接在參與癲癇發(fā)生、發(fā)展中發(fā)揮重要作用。Cx43、Cx36作為縫隙連接家族成員,在癲癇發(fā)作過程中具有不可替代的作用,本課題通過建立戊四氮誘導(dǎo)的大鼠急性癲癇模型,觀察記錄其行為學(xué)變化,檢測(cè)各組大鼠腦組織海馬和皮質(zhì)Cx43、Cx36的表達(dá)變化及其與Caspase-3、Bax、Bcl-2的關(guān)系,進(jìn)一步分析縫隙連接蛋白與凋亡的關(guān)系,探討縫隙連接蛋白與癲癇的關(guān)系,以尋找指導(dǎo)臨床藥物治療癲癇的可能靶點(diǎn)。方法:制備戊四氮(PTZ)急性大鼠癲癇模型,實(shí)驗(yàn)組采用腹腔注射PTZ,對(duì)照組腹腔注射等量生理鹽水,模型成功后隨機(jī)分為癲癇后2h、6h、12h、1d、3d組。通過HE染色、免疫組化、Western Blot、RT-PCR等方法觀察海馬和皮質(zhì)Cx43和Cx36的動(dòng)態(tài)表達(dá)變化,并且觀察Caspase-3、Bax、Bcl-2的變化以探討縫隙連接蛋白在癲癇神經(jīng)元凋亡中的可能作用機(jī)制。結(jié)果:1.免疫組織化學(xué)染色顯示,大鼠皮質(zhì)以及海馬區(qū)(CA1和CA3區(qū)顯著)可見大量Cx36陽性細(xì)胞,與對(duì)照組相比,2h起Cx36蛋白表達(dá)增多,12h達(dá)到較高水平,1-3d減弱。Cx43蛋白在2h顯著增多,之后未見明顯改變,與對(duì)照組比差異性顯著。2.Western Blot結(jié)果顯示Cx43和Cx36的變化和免疫組織化學(xué)結(jié)果一致,結(jié)果顯示Caspase-3在致癲后2h持續(xù)增高,12h-1d左右達(dá)到高峰;Bax/Bcl-2的比值在致癲后2h均顯著增加。3.PCR結(jié)果顯示,模型各組Cx43、Cx36、Caspase-3和Bax/Bcl-2比值在2h后顯著增加。4.SH-SY5Y細(xì)胞經(jīng)RA誘導(dǎo),無鎂液處理3h后,Cx43和Cx36 mRNA的變化無顯著差異。結(jié)論:1.Cx43和Cx36的變化以海馬CA1和CA3區(qū)最為明顯,Cx43和Cx36可能參與PTZ引起的急性癲癇的海馬損傷過程。2.PTZ急性癲癇大鼠的皮層和海馬區(qū)的神經(jīng)元可能通過線粒體途徑引起凋亡。
[Abstract]:Objective: epilepsy is a nervous system disease caused by abnormal discharge of neural network, which brings a great burden to individuals, families and society. Gap junction is a channel of communication between cells, carrying a lot of information exchange. Studies show that gap junction plays an important role in the occurrence and development of epilepsy. Cx43,Cx36 is a member of gap junction family. It plays an irreplaceable role in the course of epileptic seizure. In this study, we established a rat model of acute epilepsy induced by pentylenetetrazol, observed and recorded its behavioral changes, and detected Cx43, in hippocampus and cortex of rats in each group. The change of Cx36 expression and its relationship with Caspase-3,Bax,Bcl-2. The relationship between gap junction protein and apoptosis was analyzed, and the relationship between gap junction protein and epilepsy was discussed in order to find the possible target to guide clinical drug therapy for epilepsy. Methods: the acute epileptic model of pentylenetetrazole (PTZ) rats was established. The experimental group was injected intraperitoneally with normal saline by intraperitoneal injection of PTZ,. After the success of the model, the rats were randomly divided into two groups: 2 h, 6 h, 12 h and 1 d. The dynamic expression of Cx43 and Cx36 in hippocampus and cortex was observed by HE staining, immunohistochemistry and Western Blot,RT-PCR, and Caspase-3,Bax, was observed. The changes of Bcl-2 were used to investigate the possible mechanism of gap junction protein (CGN) in the apoptosis of epileptic neurons. Results: 1. Immunohistochemical staining showed that a large number of Cx36 positive cells were found in the cortex and hippocampus (CA1 and CA3) of rats. Compared with the control group, the expression of Cx36 protein increased at 2h and reached a higher level at 12h. The protein of Cx43 increased significantly at 2 h, but did not change after 2 h, which was significantly different from that of the control group. 2.Western Blot results showed that the changes of Cx43 and Cx36 were consistent with those of immunohistochemistry. The results showed that Caspase-3 increased continuously at 2 h after epilepsy, and 12h-1d reached the peak at about 2 h after epilepsy. The ratio of Bax/Bcl-2 to Bax/Bcl-2 increased significantly at 2 h after epilepsy. The results of 3.PCR showed that the ratios of Cx43,Cx36,Caspase-3 and Bax/Bcl-2 in each group were significantly increased after 2 h. 4.SH-SY5Y cells were induced by RA and treated with magnesium free solution for 3 h. There was no significant difference between Cx43 and Cx36 mRNA. Conclusion: the changes of 1.Cx43 and Cx36 in hippocampal CA1 and CA3 are the most obvious. Cx43 and Cx36 may be involved in the process of hippocampal injury induced by PTZ, and the neurons in cortex and hippocampus of 2.PTZ acute epilepsy rats may induce apoptosis through mitochondrial pathway.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R742.1

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