本文選題：魚(yú)藤酮　切入點(diǎn)：神經(jīng)細(xì)胞　出處：《南京師范大學(xué)》2014年碩士論文

【摘要】：本文運(yùn)用細(xì)胞培養(yǎng)、[Ca2+]i熒光染色、細(xì)胞凋亡DAPI染色、Western bloting等細(xì)胞學(xué)分子生物學(xué)技術(shù),綜合研究了魚(yú)藤酮誘發(fā)神經(jīng)細(xì)胞凋亡過(guò)程中,胞內(nèi)游離鈣離子([Ca2+]i)變化及其對(duì)mTOR信號(hào)通路的影響。探討了[Ca2+]i在魚(yú)藤酮致神經(jīng)細(xì)胞凋亡中的調(diào)控作用及[Ca2+]i升高機(jī)制,以及通過(guò)CaM抑制劑TFP論證了CaM在魚(yú)藤酮上調(diào)胞內(nèi)[Ca2+]i信號(hào)引發(fā)神經(jīng)細(xì)胞凋亡過(guò)程中信號(hào)轉(zhuǎn)導(dǎo)分子機(jī)制。具體結(jié)果如下： 1魚(yú)藤酮誘導(dǎo)神經(jīng)細(xì)胞[Ca2+]i升高涉及mTOR通路抑制和凋亡 以PC12和原代神經(jīng)元為對(duì)象,采用不同魚(yú)藤酮濃度(0、0.05、0.1、0.3、0.5、1μM)處理24h、或用胞內(nèi)鈣螯合劑BAPTA/AM預(yù)處理1h,然后暴露0.5和1μM魚(yú)藤酮4或24h,以Fluo-3/AM為熒光探針?lè)治黾?xì)胞內(nèi)鈣離子([Ca2+]i)的熒光強(qiáng)度,One Solution分析細(xì)胞活性,DAPI染色分析細(xì)胞凋亡,并用Western blot分析BAPTA/AM對(duì)魚(yú)藤酮誘導(dǎo)神經(jīng)細(xì)胞凋亡過(guò)程中mTOR信號(hào)通路影響。結(jié)果顯示,魚(yú)藤酮以濃度依賴的方式觸發(fā)[Ca2+]i升高導(dǎo)致神經(jīng)細(xì)胞凋亡,并關(guān)聯(lián)著mTOR及其介導(dǎo)的S6K1和4E-BP1通路抑制。BAPTA/AM可以通過(guò)阻滯mTOR通路抑制,且明顯削弱魚(yú)藤酮誘導(dǎo)的PC12細(xì)胞和原代神經(jīng)元凋亡。提示：魚(yú)藤酮誘導(dǎo)神經(jīng)細(xì)胞[Ca2+]i升高涉及mTOR通路抑制和凋亡。 2魚(yú)藤酮誘導(dǎo)[Ca2+]i升高機(jī)理及與抑制mTOR通路涉及神經(jīng)細(xì)胞凋亡關(guān)系選用內(nèi)質(zhì)網(wǎng)IP3受體抑制劑2-APB和鈣離子螯合劑EGTA來(lái)研究?jī)?nèi)質(zhì)網(wǎng)內(nèi)鈣和胞外鈣是否參與了魚(yú)藤酮誘導(dǎo)[Ca2+]i升高。PC12細(xì)胞和原代神經(jīng)元接種于6孔或96孔培養(yǎng)板中,在2-APB (100μM)或EGTA (100μM)預(yù)處理1h后,暴露0.5和1μM魚(yú)藤酮處理4或24h。分別采用One Solution分析細(xì)胞活性,以Fluo-3/AM為熒光探針?lè)治?-APB或EGTA對(duì)[Ca2+]i的影響并結(jié)合DAPI染色觀察其對(duì)魚(yú)藤酮誘發(fā)的神經(jīng)細(xì)胞凋亡的保護(hù)作用；同時(shí)利用Western Blotting檢測(cè)2-APB或EGTA阻斷胞內(nèi)鈣庫(kù)釋放或胞外鈣來(lái)源后,對(duì)神經(jīng)細(xì)胞凋亡過(guò)程mTOR信號(hào)通路的影響。結(jié)果顯示,2-APB或EGTA可以明顯降低魚(yú)藤酮誘發(fā)的[Ca2+]i水平,通過(guò)逆轉(zhuǎn)mTOR、S6K1和4E-BP1磷酸化抑制,能夠削弱魚(yú)藤酮誘導(dǎo)的PC12細(xì)胞和原代神經(jīng)元活性下降和凋亡。提示：內(nèi)質(zhì)網(wǎng)上IP3R激活,鈣釋放通道的打開(kāi),以及胞外鈣離子的進(jìn)入可能是魚(yú)藤酮引發(fā)[Ca2+]i升高的重要機(jī)制,2-APB或EGTA對(duì)神經(jīng)細(xì)胞死亡有明顯保護(hù)作用。 3魚(yú)藤酮通過(guò)[Ca2+]i升高介導(dǎo)CaM活化抑制mTOR通路涉及神經(jīng)細(xì)胞凋亡 PC12細(xì)胞和原代神經(jīng)元接種于6孔或96孔培養(yǎng)板中,在TFP (10μM)、 BAPTA/AM (30μM)、2-APB (100μM)或EGTA (100μM)預(yù)處理1h后,暴露0.5和1μM魚(yú)藤酮處理4或24h。分別采用One Solution分析細(xì)胞活性,DAPI染色分析細(xì)胞凋亡、Western Blotting檢測(cè)TFP對(duì)神經(jīng)細(xì)胞mTOR信號(hào)通路以及BAPTA/AM、2-APB、EGTA或TFP對(duì)caspase-3和PARP表達(dá)的影響。結(jié)果顯示,TFP可以明顯逆轉(zhuǎn)魚(yú)藤酮誘發(fā)的mTOR, S6K1和4E-BP1磷酸化抑制、削弱魚(yú)藤酮誘導(dǎo)的PC12細(xì)胞和原代神經(jīng)元活性下降和凋亡。魚(yú)藤酮升高[Ca2+]i介導(dǎo)caspase依賴地神經(jīng)細(xì)胞凋亡,BAPTA/AM、EGTA、2-APB或TFP阻止魚(yú)藤酮誘導(dǎo)原代神經(jīng)元caspase通路激活。提示：魚(yú)藤酮通過(guò)[Ca2+]i升高介導(dǎo)CaM活化抑制mTOR通路涉及神經(jīng)細(xì)胞凋亡。
[Abstract]:Using cell culture, [Ca2+]i staining, apoptosis DAPI staining, Western bloting cytology and molecular biology techniques, a comprehensive study of the process of nerve cell apoptosis induced by rotenone, intracellular free calcium ([Ca2+]i) changes and the effect of mTOR signal pathway. To investigate the regulatory role of [Ca2+]i and [Ca2+]i in rotenone induced apoptosis of nerve cells the mechanism of elevated, and the CaM inhibitor TFP demonstrated CaM [Ca2+]i in rotenone upregulated intracellular signal triggered signal transduction apoptosis mechanism. The main results are as follows:
1 the increase of [Ca2+]i induced by rotenone involves the inhibition and apoptosis of mTOR pathway
With PC12 and primary neurons as the object, using different concentrations of rotenone (0,0.05,0.1,0.3,0.5,1 M) 24h, or by intracellular calcium chelator BAPTA/AM 1H pretreatment, and then exposed to 0.5 and 1 M rotenone 4 or 24h, using Fluo-3/AM as a calcium fluorescent probe analysis ([Ca2+]i) fluorescence intensity, cell analysis the activity of One Solution on cell apoptosis DAPI staining and Western blot analysis of BAPTA/AM mTOR signal pathway induced apoptosis of rotenone. The results showed that rotenone in a concentration dependent manner to trigger increased [Ca2+]i induced apoptosis of nerve cells, and associated with the S6K1 and 4E-BP1 pathway and mTOR mediated inhibition of.BAPTA/AM can be inhibited by blockage of the mTOR pathway, and significantly weaken the rotenone induced PC12 cells and primary neurons apoptosis induced by rotenone. Tip: nerve cells increased [Ca2+]i involves mTOR pathway inhibition and Apoptosis.
2 rotenone induced [Ca2+]i increase mechanism and inhibition of mTOR pathway involved in endoplasmic reticulum and IP3 receptor inhibitor 2-APB and calcium chelator EGTA the relationship between the apoptosis of nerve cell is chosen to study the endoplasmic reticulum calcium and extracellular calcium in rotenone induced increase of [Ca2+]i.PC12 cells and primary neurons cultured in a 6 or 96 hole culture plate. In the 2-APB (100 M) or EGTA (100 M) 1H pretreatment after exposure to 0.5 and 1 M rotenone treatment 4 or 24h. respectively by One Solution analysis of cells with Fluo-3/AM fluorescence probe to analyze the effect of 2-APB or EGTA on [Ca2+]i and DAPI staining to observe the protective effect of rotenone induced nerve cell apoptosis; at the same time using Western Blotting to detect 2-APB or EGTA blocking intracellular calcium release or extracellular calcium sources, effects on the apoptosis of the mTOR signaling pathway. The results showed that 2-APB or EGTA can be Decreased rotenone induced [Ca2+]i levels by reverse mTOR, S6K1 and 4E-BP1 phosphorylation inhibition can weaken the rotenone induced PC12 cells and primary neuronal activity decreased and apoptosis. Tip: endoplasmic reticulum IP3R activation, open the calcium release channel, and extracellular calcium entry may be an important mechanism of rotenone increased [Ca2+]i the 2-APB or EGTA has obvious protective effect on neuronal cell death.
3 rotenone mediated CaM activation inhibition of mTOR pathway involved in neuronal apoptosis through [Ca2+]i
PC12 cells and primary neurons cultured in a 6 or 96 hole culture plate, TFP (10 M), BAPTA/AM (30 M), 2-APB (100 M) or EGTA (100 M) 1H pretreatment after exposure to 0.5 and 1 M rotenone treatment respectively using One 4 or 24h. analysis of Solution cell activity, DAPI staining analysis of cell apoptosis, Western Blotting detection of TFP on nerve cell mTOR signaling pathway and BAPTA/AM, 2-APB, EGTA or TFP influence on the expression of Caspase-3 and PARP. The results showed that TFP can significantly reverse the rotenone induced mTOR S6K1 and inhibition of 4E-BP1 phosphorylation, weaken the rotenone induced PC12 cells and the primary neuronal activity decreased and apoptosis increased. Rotenone [Ca2+]i mediated caspase dependent apoptosis, neural cell BAPTA/AM, EGTA, 2-APB or TFP prevented rotenone induced primary neuronal activation of caspase signaling pathway. Note: [Ca2+]i increased by rotenone activated CaM mediated inhibition of mTOR pathway involved And apoptosis of nerve cells.

【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.5

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相關(guān)期刊論文 前1條

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本文編號(hào)：1677758