【摘要】： 目的:探討線(xiàn)粒體氯通道/細(xì)胞內(nèi)氯通道4(mtCLIC/CLIC4)在H_2O_2誘導(dǎo)的大鼠膠質(zhì)細(xì)胞損傷過(guò)程中作用及機(jī)制。 方法:根據(jù)RNAi設(shè)計(jì)原理以及基因重組技術(shù)構(gòu)建siRNA-CLIC4表達(dá)載體,轉(zhuǎn)染SD大鼠C6膠質(zhì)瘤細(xì)胞,對(duì)比觀(guān)察CLIC4和CLIC4-RNAi在H_2O_2誘導(dǎo)的大鼠膠質(zhì)細(xì)胞損傷過(guò)程中的作用。 結(jié)果:(1)H_2O_2誘導(dǎo)的大鼠C6細(xì)胞損傷過(guò)程中,CLIC4蛋白表達(dá)顯著增強(qiáng),同時(shí)Caspase3、VDAC1蛋白表達(dá)明顯增強(qiáng)以及Bax/Bcl-2比值明顯增高。(2)首次完成大鼠C6膠質(zhì)瘤細(xì)胞CLIC4cDNA釣取,亞克隆和測(cè)序,發(fā)現(xiàn)新的SD大鼠膠質(zhì)細(xì)胞CLIC4序列,將結(jié)果登錄到GenBank,獲得登錄號(hào):Bankit878161,EF397567。(3)首次利用RNAi技術(shù)成功構(gòu)建siRNA-CLIC4表達(dá)質(zhì)粒,顯著抑制CLIC4基因表達(dá),并獲得穩(wěn)定轉(zhuǎn)染C6細(xì)胞株。(4)在CLIC4干涉組(pSH1Si-CLIC4)中盡管總CLIC4蛋白表達(dá)在RNAi作用下明顯下調(diào),但伴隨H_2O_2濃度的增加,細(xì)胞核內(nèi)的CLIC4蛋白表達(dá)仍顯著增加,促進(jìn)了H_2O_2誘導(dǎo)的細(xì)胞凋亡。 結(jié)論:發(fā)現(xiàn)新的SD大鼠膠質(zhì)細(xì)胞CLIC4cDNA的基因序列并成功構(gòu)建siRNA-CLIC4有效表達(dá)質(zhì)粒及穩(wěn)定轉(zhuǎn)染的C6細(xì)胞株。在H_2O_2誘導(dǎo)的C6細(xì)胞損傷過(guò)程中,CLIC4作為凋亡效應(yīng)分子參與了其線(xiàn)粒體途徑的凋亡過(guò)程。而通過(guò)RNAi技術(shù)下調(diào)CLIC4表達(dá)后,同樣明顯促進(jìn)了H_2O_2誘導(dǎo)的細(xì)胞凋亡,該過(guò)程中細(xì)胞核靶向的CLIC4大量入核,可能與CLIC4作為離子通道影響了細(xì)胞核內(nèi)離子平衡并改變其pH值水平有關(guān),進(jìn)而通過(guò)pH-依賴(lài)的核酸內(nèi)切酶激活的細(xì)胞核凋亡途徑加速細(xì)胞凋亡。 本研究為探討細(xì)胞內(nèi)氯通道在神經(jīng)退行性疾病發(fā)病過(guò)程中所發(fā)揮的作用進(jìn)行了新的探索,也為尋找腫瘤基因治療新的作用靶點(diǎn)進(jìn)行了有益的嘗試。
[Abstract]:Aim: to investigate the role and mechanism of mitochondrial chloride channel / intracellular chloride channel 4 (mtCLIC/CLIC4) in H_2O_2 induced glial injury in rats. Methods: according to the principle of RNAi design and gene recombination, the siRNA-CLIC4 expression vector was constructed and transfected into SD rat C6 glioma cells. The role of CLIC4 and CLIC4-RNAi in the process of H_2O_2 induced glial cell injury was observed. Results: (1) during the injury of C6 cells induced by H_2O_2, the expression of CLIC4 protein was significantly increased, and the expression of Caspase3, was also significantly increased. The expression of VDAC1 protein and the ratio of Bax/Bcl-2 were significantly increased. (2) A novel CLIC4 sequence of rat glial cells of SD was found after CLIC4cDNA fishing, subcloning and sequencing of rat C6 glioma cells were completed for the first time, and the results were logged into GenBank,. Accession number: Bankit878161,EF397567. (3) successfully constructed siRNA-CLIC4 expression plasmid by RNAi technique for the first time, and significantly inhibited the expression of CLIC4 gene. Stable transfection of C6 cell line was obtained. (4) in CLIC4 interference group (pSH1Si-CLIC4), although the expression of total CLIC4 protein was significantly down-regulated by RNAi, the expression of CLIC4 protein in the nucleus was still significantly increased with the increase of H_2O_2 concentration. H_2O_2-induced apoptosis was promoted. Conclusion: the new gene sequence of CLIC4cDNA in glial cells of SD rats was found and the effective expression plasmid of siRNA-CLIC4 and the stable transfected C6 cell line were successfully constructed. In the process of C6 cell injury induced by H_2O_2, CLIC4 is involved in the apoptosis process of mitochondria pathway as an effector of apoptosis. After down-regulation of CLIC4 expression by RNAi technique, apoptosis induced by H_2O_2 was also promoted, during which a large number of nuclear targeted CLIC4 entered the nucleus. It may be related to the effect of CLIC4 as an ion channel on the ion balance in the nucleus and to change the level of pH, and then accelerate the apoptosis through the pathway of nucleus apoptosis activated by pH- dependent endonuclease. In order to explore the role of intracellular chloride channels in the pathogenesis of neurodegenerative diseases, we have made a new attempt to find new targets for tumor gene therapy.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 張海寧;血管性認(rèn)知障礙大鼠腦突觸小體的差異蛋白質(zhì)組學(xué)研究[D];吉林大學(xué);2011年

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本文編號(hào)：2406053