本文選題：C6細(xì)胞　切入點(diǎn)：雙氫睪酮　出處：《復(fù)旦大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景年齡相關(guān)的雄激素水平下降與多種神經(jīng)退行性疾病相關(guān),包括阿爾茨海默病海默病(Alzheimer's Disease,AD)、焦慮抑郁癥、腦萎縮等。越來越多資料提示,雙氫睪酮(Dihydrotestosterone, DHT)在此類疾病的預(yù)防與治療中具有廣泛的神經(jīng)生物學(xué)作用,有助于抑制疾病病理損傷、修復(fù)損傷的神經(jīng)元、突觸可塑性修復(fù)等神經(jīng)保護(hù)效應(yīng)。目前關(guān)于雄激素神經(jīng)保護(hù)作用機(jī)制研究較少,主要包括： (1)調(diào)節(jié)腦內(nèi)β-淀粉樣蛋白釋放； (2)抗氧化應(yīng)激： (3)抗凋亡作用；(4)促進(jìn)神經(jīng)元生長、分化。進(jìn)一步闡明雄激素的神經(jīng)保護(hù)作用及其分子作用機(jī)制,可能為我們探索預(yù)防與治療神經(jīng)退行性疾病的新方法提供非常重要的理論與臨床依據(jù)。研究目的本研究采用C6細(xì)胞作為體外神經(jīng)膠質(zhì)細(xì)胞研究模型,利用U18666A誘發(fā)C6細(xì)胞凋亡活動(dòng)、LY294002抑制PI3K/Akt信號(hào)通路,觀察PI3K/Akt信號(hào)通路在雙氫睪酮(DHT)抗U18666A誘導(dǎo)的C6細(xì)胞凋亡活動(dòng)的作用及其分子機(jī)制。從而進(jìn)一步探討PI3K/Akt信號(hào)通路在雄激素抗U18666A誘發(fā)C6細(xì)胞凋亡作用中的意義。研究方法第一部分通過AnnexinV-FITC/PI雙染流式細(xì)胞技術(shù)、Hochest33342染色檢測(cè)法,分別定量和定性檢測(cè)不同濃度的U18666A對(duì)C6細(xì)胞凋亡活動(dòng)的形態(tài)學(xué)變化影響。第二部分通過AnnexinV-FITC/PI雙染流式細(xì)胞術(shù)、Hochest33342染色檢測(cè)法從形態(tài)學(xué)上觀察DHT對(duì)U18666A誘導(dǎo)C6細(xì)胞凋亡的保護(hù)作用,以及LY294002對(duì)DHT凋亡保護(hù)的阻斷作用。第三部分利用Western Blot檢測(cè)法,觀察DHT對(duì)Akt磷酸化激活作用,以及LY294002對(duì)DHT的Akt磷酸化激活作用的影響。此外,我們還進(jìn)一步利用Western Blot檢測(cè)法定量觀察DHT、LY294002對(duì)凋亡相關(guān)蛋白Seladin-1、Bcl-2家族、IAP家族、Caspase-3表達(dá)變化的影響,探討PI3K/Akt信號(hào)通路及其下游凋亡相關(guān)分子Seladin-1、BCL-XL、Bax、Survivin、Caspase-3表達(dá)的變化在雄激素抗U18666A凋亡作用中的分子機(jī)制。研究結(jié)果第一部分免疫熒光檢測(cè)發(fā)現(xiàn),在熒光顯微鏡微鏡下發(fā)現(xiàn)U18666A能顯著誘導(dǎo)C6細(xì)胞凋亡發(fā)生,凋亡細(xì)胞數(shù)量明顯增加。通過AnnexinV-FITC/PI雙染流式細(xì)胞技術(shù)定量檢測(cè)發(fā)現(xiàn),相較空白對(duì)照組,不同濃度(0.5μg/ml,1.0μg/ml,2.5μg/ml)的U18666A處理能夠顯著增加C6細(xì)胞的總凋亡率(P0.05)。上述結(jié)果提示,不同濃度的U18666A(實(shí)驗(yàn)采用)能夠顯著誘導(dǎo)C6膠質(zhì)細(xì)胞發(fā)生凋亡活動(dòng)。第二部分為了觀察DHT對(duì)U18666A誘導(dǎo)C6膠質(zhì)細(xì)胞亡活動(dòng)的形態(tài)學(xué)影響,我們用DHT(10-2μM)預(yù)處理1h后給予U18666A(1.0μg/ml)刺激48h,在熒光顯微鏡下發(fā)現(xiàn)C6細(xì)胞凋亡細(xì)胞數(shù)量明顯減少,而P13K抑制劑LY294002(50 μM)預(yù)處理2h后發(fā)現(xiàn)DHT的抗凋亡作用明顯被抑制。此外,AnnexinV-FITC/PI雙染流式細(xì)胞檢測(cè)發(fā)現(xiàn),與U18666A處理組相比,DHT預(yù)處理可以明顯降低U18666A誘導(dǎo)的C6細(xì)胞總凋亡率(P0.05),而P13K抑制劑LY294002(50μM)可以抑制DHT的抗凋亡活動(dòng)(P0.05)。上述結(jié)果提示,DHT可以明顯阻斷U18666A誘導(dǎo)的C6細(xì)胞凋亡活動(dòng),而P13K阻斷劑可以明顯抑制DHT的抗凋亡保護(hù)作用。第三部分首先我們利用Western Blot檢測(cè)發(fā)現(xiàn),與U18666A處理組比較,DHT處理后C6細(xì)胞的磷酸化Akt表達(dá)量顯著升高(P0.05),而LY294002預(yù)處理后能夠顯著抑制調(diào)DHT誘導(dǎo)的磷酸化Akt水平升高(P0.05)。上述結(jié)果提示,DHT可以明顯激活C6細(xì)胞的Akt磷酸化活動(dòng),而P13K阻斷劑可以明顯抑制DHT的Akt激活效應(yīng)。此外,我們進(jìn)一步利用Western Blot檢測(cè)發(fā)現(xiàn)：1)與空白組比較,U18666A可以明顯下調(diào)凋亡保護(hù)蛋白Seladin-1表達(dá)(P0.05),上調(diào)促凋亡蛋白Caspase-3表達(dá)(P0.05)：2)與U18666A處理組對(duì)比發(fā)現(xiàn),DHT預(yù)處理后可以明顯上調(diào)凋亡保護(hù)蛋白Seladin-1、Survivin、BCL-XL表達(dá)(P0.05),下調(diào)促凋亡蛋白Caspase-3、Bax表達(dá)(P0.05)。3)與DHT處理組(DHT+U18666A)比較,P13K阻斷劑LY294002可以明顯抑制DHT誘導(dǎo)的促凋亡蛋白Caspase-3、Bax下調(diào)表達(dá)(P0.05),以及抑制DHT誘導(dǎo)的Seladin-1、Survivin、BCL-XL上調(diào)表達(dá)(P0.05)。研究結(jié)論根據(jù)上述實(shí)驗(yàn)結(jié)果,在C6細(xì)胞模型上,我們發(fā)現(xiàn)：1)從凋亡形態(tài)學(xué)上我們發(fā)現(xiàn),不同濃度的U18666A均可以明顯誘導(dǎo)體外培養(yǎng)的C6細(xì)胞發(fā)生凋亡現(xiàn)象。上述結(jié)果提示,U18666A對(duì)C6膠質(zhì)細(xì)胞具有促凋亡作用。2)從凋亡形態(tài)學(xué)上我們發(fā)現(xiàn),DHT具有明顯的抗U18666A誘導(dǎo)的C6細(xì)胞凋亡效應(yīng),而P13K抑制劑LY294002可以明顯阻斷DHT抗凋亡保護(hù)作用,我們的結(jié)果證明,PI3K/Akt信號(hào)通路參與了DHT抗U18666A誘導(dǎo)的C6細(xì)胞凋亡效應(yīng)作用。3)此外,我們發(fā)現(xiàn)DHT可以顯著上調(diào)凋亡保護(hù)蛋白Seladin-1、BCL-XL、 Survivin表達(dá),下調(diào)促凋亡蛋白Bax、Caspase-3表達(dá)發(fā)揮抗凋亡保護(hù)作用,而DHT上述效應(yīng)可以明顯被P13K抑制劑LY294002所阻斷。因此,我們推測(cè)DHT可能通過激活PI3K/Akt信號(hào)通路調(diào)控下游的凋亡相關(guān)蛋白Seladin-1、Survivin、Caspase-3、BCL-XL、Bax表達(dá),從而發(fā)揮抗凋亡保護(hù)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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741

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