鱼藤酮通过钙信号转导抑制mTOR通路诱导神经细胞凋亡机理研究

发布时间：2018-03-28 19:28

本文选题：鱼藤酮　切入点：神经细胞　出处：《南京师范大学》2014年硕士论文

【摘要】：本文运用细胞培养、[Ca2+]i荧光染色、细胞凋亡DAPI染色、Western bloting等细胞学分子生物学技术,综合研究了鱼藤酮诱发神经细胞凋亡过程中,胞内游离钙离子([Ca2+]i)变化及其对mTOR信号通路的影响。探讨了[Ca2+]i在鱼藤酮致神经细胞凋亡中的调控作用及[Ca2+]i升高机制,以及通过CaM抑制剂TFP论证了CaM在鱼藤酮上调胞内[Ca2+]i信号引发神经细胞凋亡过程中信号转导分子机制。具体结果如下： 1鱼藤酮诱导神经细胞[Ca2+]i升高涉及mTOR通路抑制和凋亡以PC12和原代神经元为对象,采用不同鱼藤酮浓度(0、0.05、0.1、0.3、0.5、1μM)处理24h、或用胞内钙螯合剂BAPTA/AM预处理1h,然后暴露0.5和1μM鱼藤酮4或24h,以Fluo-3/AM为荧光探针分析细胞内钙离子([Ca2+]i)的荧光强度,One Solution分析细胞活性,DAPI染色分析细胞凋亡,并用Western blot分析BAPTA/AM对鱼藤酮诱导神经细胞凋亡过程中mTOR信号通路影响。结果显示,鱼藤酮以浓度依赖的方式触发[Ca2+]i升高导致神经细胞凋亡,并关联着mTOR及其介导的S6K1和4E-BP1通路抑制。BAPTA/AM可以通过阻滞mTOR通路抑制,且明显削弱鱼藤酮诱导的PC12细胞和原代神经元凋亡。提示：鱼藤酮诱导神经细胞[Ca2+]i升高涉及mTOR通路抑制和凋亡。 2鱼藤酮诱导[Ca2+]i升高机理及与抑制mTOR通路涉及神经细胞凋亡关系选用内质网IP3受体抑制剂2-APB和钙离子螯合剂EGTA来研究内质网内钙和胞外钙是否参与了鱼藤酮诱导[Ca2+]i升高。PC12细胞和原代神经元接种于6孔或96孔培养板中,在2-APB (100μM)或EGTA (100μM)预处理1h后,暴露0.5和1μM鱼藤酮处理4或24h。分别采用One Solution分析细胞活性,以Fluo-3/AM为荧光探针分析2-APB或EGTA对[Ca2+]i的影响并结合DAPI染色观察其对鱼藤酮诱发的神经细胞凋亡的保护作用；同时利用Western Blotting检测2-APB或EGTA阻断胞内钙库释放或胞外钙来源后,对神经细胞凋亡过程mTOR信号通路的影响。结果显示,2-APB或EGTA可以明显降低鱼藤酮诱发的[Ca2+]i水平,通过逆转mTOR、S6K1和4E-BP1磷酸化抑制,能够削弱鱼藤酮诱导的PC12细胞和原代神经元活性下降和凋亡。提示：内质网上IP3R激活,钙释放通道的打开,以及胞外钙离子的进入可能是鱼藤酮引发[Ca2+]i升高的重要机制,2-APB或EGTA对神经细胞死亡有明显保护作用。 3鱼藤酮通过[Ca2+]i升高介导CaM活化抑制mTOR通路涉及神经细胞凋亡 PC12细胞和原代神经元接种于6孔或96孔培养板中,在TFP (10μM)、 BAPTA/AM (30μM)、2-APB (100μM)或EGTA (100μM)预处理1h后,暴露0.5和1μM鱼藤酮处理4或24h。分别采用One Solution分析细胞活性,DAPI染色分析细胞凋亡、Western Blotting检测TFP对神经细胞mTOR信号通路以及BAPTA/AM、2-APB、EGTA或TFP对caspase-3和PARP表达的影响。结果显示,TFP可以明显逆转鱼藤酮诱发的mTOR, S6K1和4E-BP1磷酸化抑制、削弱鱼藤酮诱导的PC12细胞和原代神经元活性下降和凋亡。鱼藤酮升高[Ca2+]i介导caspase依赖地神经细胞凋亡,BAPTA/AM、EGTA、2-APB或TFP阻止鱼藤酮诱导原代神经元caspase通路激活。提示：鱼藤酮通过[Ca2+]i升高介导CaM活化抑制mTOR通路涉及神经细胞凋亡。
[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.

【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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