雄激素抗U18666A诱导细胞凋亡的保护作用研究

发布时间：2018-03-07 11:10

本文选题：C6细胞　切入点：双氢睾酮　出处：《复旦大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景年龄相关的雄激素水平下降与多种神经退行性疾病相关,包括阿尔茨海默病海默病(Alzheimer's Disease,AD)、焦虑抑郁症、脑萎缩等。越来越多资料提示,双氢睾酮(Dihydrotestosterone, DHT)在此类疾病的预防与治疗中具有广泛的神经生物学作用,有助于抑制疾病病理损伤、修复损伤的神经元、突触可塑性修复等神经保护效应。目前关于雄激素神经保护作用机制研究较少,主要包括： (1)调节脑内β-淀粉样蛋白释放； (2)抗氧化应激： (3)抗凋亡作用；(4)促进神经元生长、分化。进一步阐明雄激素的神经保护作用及其分子作用机制,可能为我们探索预防与治疗神经退行性疾病的新方法提供非常重要的理论与临床依据。研究目的本研究采用C6细胞作为体外神经胶质细胞研究模型,利用U18666A诱发C6细胞凋亡活动、LY294002抑制PI3K/Akt信号通路,观察PI3K/Akt信号通路在双氢睾酮(DHT)抗U18666A诱导的C6细胞凋亡活动的作用及其分子机制。从而进一步探讨PI3K/Akt信号通路在雄激素抗U18666A诱发C6细胞凋亡作用中的意义。研究方法第一部分通过AnnexinV-FITC/PI双染流式细胞技术、Hochest33342染色检测法,分别定量和定性检测不同浓度的U18666A对C6细胞凋亡活动的形态学变化影响。第二部分通过AnnexinV-FITC/PI双染流式细胞术、Hochest33342染色检测法从形态学上观察DHT对U18666A诱导C6细胞凋亡的保护作用,以及LY294002对DHT凋亡保护的阻断作用。第三部分利用Western Blot检测法,观察DHT对Akt磷酸化激活作用,以及LY294002对DHT的Akt磷酸化激活作用的影响。此外,我们还进一步利用Western Blot检测法定量观察DHT、LY294002对凋亡相关蛋白Seladin-1、Bcl-2家族、IAP家族、Caspase-3表达变化的影响,探讨PI3K/Akt信号通路及其下游凋亡相关分子Seladin-1、BCL-XL、Bax、Survivin、Caspase-3表达的变化在雄激素抗U18666A凋亡作用中的分子机制。研究结果第一部分免疫荧光检测发现,在荧光显微镜微镜下发现U18666A能显著诱导C6细胞凋亡发生,凋亡细胞数量明显增加。通过AnnexinV-FITC/PI双染流式细胞技术定量检测发现,相较空白对照组,不同浓度(0.5μg/ml,1.0μg/ml,2.5μg/ml)的U18666A处理能够显著增加C6细胞的总凋亡率(P0.05)。上述结果提示,不同浓度的U18666A(实验采用)能够显著诱导C6胶质细胞发生凋亡活动。第二部分为了观察DHT对U18666A诱导C6胶质细胞亡活动的形态学影响,我们用DHT(10-2μM)预处理1h后给予U18666A(1.0μg/ml)刺激48h,在荧光显微镜下发现C6细胞凋亡细胞数量明显减少,而P13K抑制剂LY294002(50 μM)预处理2h后发现DHT的抗凋亡作用明显被抑制。此外,AnnexinV-FITC/PI双染流式细胞检测发现,与U18666A处理组相比,DHT预处理可以明显降低U18666A诱导的C6细胞总凋亡率(P0.05),而P13K抑制剂LY294002(50μM)可以抑制DHT的抗凋亡活动(P0.05)。上述结果提示,DHT可以明显阻断U18666A诱导的C6细胞凋亡活动,而P13K阻断剂可以明显抑制DHT的抗凋亡保护作用。第三部分首先我们利用Western Blot检测发现,与U18666A处理组比较,DHT处理后C6细胞的磷酸化Akt表达量显著升高(P0.05),而LY294002预处理后能够显著抑制调DHT诱导的磷酸化Akt水平升高(P0.05)。上述结果提示,DHT可以明显激活C6细胞的Akt磷酸化活动,而P13K阻断剂可以明显抑制DHT的Akt激活效应。此外,我们进一步利用Western Blot检测发现：1)与空白组比较,U18666A可以明显下调凋亡保护蛋白Seladin-1表达(P0.05),上调促凋亡蛋白Caspase-3表达(P0.05)：2)与U18666A处理组对比发现,DHT预处理后可以明显上调凋亡保护蛋白Seladin-1、Survivin、BCL-XL表达(P0.05),下调促凋亡蛋白Caspase-3、Bax表达(P0.05)。3)与DHT处理组(DHT+U18666A)比较,P13K阻断剂LY294002可以明显抑制DHT诱导的促凋亡蛋白Caspase-3、Bax下调表达(P0.05),以及抑制DHT诱导的Seladin-1、Survivin、BCL-XL上调表达(P0.05)。研究结论根据上述实验结果,在C6细胞模型上,我们发现：1)从凋亡形态学上我们发现,不同浓度的U18666A均可以明显诱导体外培养的C6细胞发生凋亡现象。上述结果提示,U18666A对C6胶质细胞具有促凋亡作用。2)从凋亡形态学上我们发现,DHT具有明显的抗U18666A诱导的C6细胞凋亡效应,而P13K抑制剂LY294002可以明显阻断DHT抗凋亡保护作用,我们的结果证明,PI3K/Akt信号通路参与了DHT抗U18666A诱导的C6细胞凋亡效应作用。3)此外,我们发现DHT可以显著上调凋亡保护蛋白Seladin-1、BCL-XL、 Survivin表达,下调促凋亡蛋白Bax、Caspase-3表达发挥抗凋亡保护作用,而DHT上述效应可以明显被P13K抑制剂LY294002所阻断。因此,我们推测DHT可能通过激活PI3K/Akt信号通路调控下游的凋亡相关蛋白Seladin-1、Survivin、Caspase-3、BCL-XL、Bax表达,从而发挥抗凋亡保护作用。
[Abstract]:On the background of the age related decline in androgen levels associated with many neurodegenerative diseases, including Alzheimer's disease, Alzheimer's disease (Alzheimer's, Disease, AD), anxiety depression, brain atrophy. More and more data suggest that dihydrotestosterone (Dihydrotestosterone, DHT) has extensive neurobiological effects in the prevention and treatment of such diseases, help inhibition of pathological damage, repair of damaged neurons, synaptic plasticity and repair of nerve protective effect. The androgen neuroprotective mechanism of study mainly includes: (1) to regulate the release of beta amyloid in the brain; (2) oxidative stress: (3) the anti apoptosis effect; (4) to promote the growth of neurons differentiation. To further elucidate the mechanism, neuroprotective effect and molecular androgen, may provide for us to explore new methods of prevention and treatment of neurodegenerative diseases A very important theoretical and clinical basis. The purpose of this study using C6 cells as an in vitro model of glial cells, U18666A induced apoptosis of C6 cells by LY294002, inhibition of the PI3K/Akt signaling pathway, PI3K/Akt signaling pathway in the observation of dihydrotestosterone (DHT) effect and molecular mechanism of anti U18666A induced apoptosis of C6 cells in order to further explore the PI3K/Akt activities. The signal pathway in U18666A induced apoptosis in C6 cells, anti androgen effect significance. Methods: the first part by AnnexinV-FITC/PI double staining flow cytometry, Hochest33342 staining assay, the morphological changes were quantitative and qualitative detection of different concentrations of U18666A on C6 cell apoptosis activity. The effects of the second part of the AnnexinV-FITC/PI double staining flow cytometry. To observe the protection of DHT on U18666A induced apoptosis of C6 cells in morphology Hochest33342 staining method Effect of LY294002 on apoptosis of DHT, and the protection of the blocking effect. The third part is the use of Western Blot method, observe the DHT activation of Akt phosphorylation, and impact of Akt LY294002 on the DHT of phosphoric acid activation. In addition, we also use Western Blot method to detect the quantitative observation of DHT, LY294002 on apoptosis related protein Seladin-1, Bcl-2 family, IAP family, influence the expression of Caspase-3 on PI3K/Akt signaling pathway and its downstream apoptosis related molecules Seladin-1, BCL-XL, Bax, Survivin, molecular mechanism of the expression of Caspase-3 in the anti apoptosis effect of androgen U18666A. The research results of the first part of immunofluorescence detected under fluorescence microscope microscope showed that U18666A significantly induced C6 cell apoptosis, the number of apoptotic cells increased significantly. By AnnexinV-FITC/PI double staining and flow cytometry were used to detect, compared to the blank Control group, different concentration (0.5 g/ml, 1 g/ml, 2.5 g/ml) U18666A treatment could significantly increase the total apoptosis rate of C6 cells (P0.05). The results indicated that different concentrations of U18666A (Experiment) could significantly induce C6 apoptosis of glial cells. The second part in order to observe the effect of DHT on the morphological effect U18666A induced C6 cell apoptosis activity, we used DHT (10-2 M) pretreatment of 1H treated with U18666A (1 g/ml) stimulated 48h under fluorescence microscope and found that the number of apoptosis of C6 cells significantly decreased, while the P13K inhibitor LY294002 (50 M) 2h after pretreatment of the anti apoptotic effect of DHT was found suppression is obvious. In addition, AnnexinV-FITC/PI double staining detected by flow cytometry, compared with U18666A group, DHT pretreatment can decrease U18666A induced C6 cell apoptosis rate (P0.05), and P13K inhibitor LY294002 (50 M) can inhibit the anti apoptotic activity (DHT P0.05). These results suggest that DHT can markedly inhibit C6 induced apoptosis of U18666A cells, and P13K blocked the protective effect of anti apoptotic agent can obviously inhibit DHT. In the third part, we use the Western Blot detection showed that compared with U18666A treatment, after the treatment of DHT phosphorylation of Akt C6 cells expression increased significantly (P0.05), and LY294002 pretreatment can significantly inhibit the phosphorylation level of Akt increased regulation induced by DHT (P0.05). The results suggest that the phosphorylation of Akt DHT can significantly activate C6 cells, and the effect of blocking agent P13K can significantly inhibit DHT activation of Akt. In addition, we further use of Western Blot detected: 1) compared with the control group, U18666A can obviously down regulate the expression of apoptosis protein Seladin-1 (P0.05), up-regulated the expression of apoptotic protein Caspase-3 (P0.05):2) and U18666A treatment group comparison, after DHT pretreatment can Increased apoptosis protein Seladin-1, Survivin, BCL-XL (P0.05) expression, down-regulation of the pro apoptotic protein Caspase-3, expression of Bax (P0.05).3) and DHT treatment group (DHT+U18666A), P13K blocking Pro apoptotic protein Caspase-3 LY294002 could significantly inhibit DHT induced down-regulation of Bax expression, (P0.05), and Seladin-1, inhibition of DHT the induction of Survivin, BCL-XL expression (P0.05). Conclusion according to the above results, the C6 cell model, we found that: 1) from apoptosis morphology we found that different concentrations of U18666A can induce C6 cells apoptosis. These results suggest that U18666A may induce apoptosis of.2 C6 glial cells from apoptosis morphology) we found that DHT has obvious anti U18666A induced C6 cell apoptosis effect, while the P13K inhibitor LY294002 can significantly block the protective effect of DHT against apoptosis, we The results show that PI3K/Akt signaling pathway is involved in the effect of C6 cell apoptosis induced by anti DHT U18666A.3) in addition, we found that DHT can significantly increase apoptosis protein Seladin-1, BCL-XL, Survivin expression, down-regulation of the pro apoptotic protein Bax, Caspase-3 expression of anti apoptosis play a protective effect, and the effect of DHT was blocked by P13K inhibitors of LY294002. Therefore, we speculate that DHT may activate downstream PI3K/Akt signaling pathway of apoptosis related protein Seladin-1, Survivin, Caspase-3, BCL-XL, Bax expression, to play the anti apoptotic protective effect.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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