AMPK-mTOR通路在锰诱导PC12细胞自噬中的作用研究
发布时间：2018-06-21 19:14
本文选题：AMPK + mTOR ； 参考：《广西医科大学》2017年硕士论文
【摘要】：目的:自噬是真核细胞中降解长寿命蛋白和调节细胞器代谢的主要机制,受到多种信号通路的调控。本研究以锰诱导PC12细胞建立锰中毒性帕金森综合征体外细胞模型,探讨AMPK-mTOR信号通路在锰诱导PC12细胞自噬中的作用,为锰中毒性帕金森综合征的发病机制及药物研究提供思路。方法:根据不同浓度及不同作用时间锰对PC12细胞存活率的影响,选择合适的锰浓度及作用时间,建立锰中毒性帕金森综合征体外细胞模型,再给予Compound C进行干预:CCK-8法检测细胞存活率,透射电子显微镜观察PC12细胞经锰处理后的超微结构变化,分子水平检测自噬标记蛋白LC3及AMPK-mTOR通路关键蛋白p-AMPK、p-mTOR、p-4E-BP1、p-p70S6K的表达变化。结果:1、PC12细胞在不同锰浓度下分别作用24h、48h、72h,用CCK-8法检测计算细胞存活率,染锰组与对照组间比较,P0.01,不同染锰组间比较,P0.01,同一染锰组在不同时间点间比较,P0.01,差异均具有显著性。2、PC12细胞按Control组、Mn组、CC组、Mn+CC组分别加药处理24h,CCK-8法检测计算Control组细胞存活率为(100.00±0)%,Mn组、CC组、Mn+CC组细胞存活率均较Control组下降,P0.01,差异有显著性,而Mn组与Mn+CC组相比,Mn+CC组细胞存活率较大,P0.05,差异有统计学意义。3、PC12细胞经锰处理后,LC3-Ⅱ/LC3-Ⅰ表达量增加,呈浓度和时间依赖性(P㩳0.01)。4、透射电子显微镜下可见染锰组细胞内自噬体及自噬溶酶体增加。5、与对照组相比,Mn组PC12细胞中p-AMPK、LC3-Ⅱ/LC3-Ⅰ蛋白的相对表达量明显增多(P㩳0.01),p-mTOR、p-p70S6K、p-4E-BP1蛋白的相对表达量显著减少(P㩳0.01);而Mn+CC组中p-AMPK、p-mTOR、p-p70S6K、p-4E-BP1、LC3-Ⅱ/LC3-Ⅰ的表达量均介于Control组与Mn组之间(P㩳0.05)。结论:1、锰能诱导PC12细胞激活自噬,抑制细胞增殖,且呈浓度和时间依赖性。2、Compound C可抑制锰对PC12细胞自噬的作用。3、锰可能通过AMPK-mTOR信号通路诱导PC12细胞激活自噬,抑制细胞增殖。4、AMPK-mTOR信号通路可能参与锰中毒性帕金森综合征的发病机制。
[Abstract]:Aim: autophagy is the main mechanism of degradation of long-lived protein and regulation of organelle metabolism in eukaryotic cells, which is regulated by multiple signaling pathways. In this study, we used manganese to induce PC12 cells to establish the model of manganese toxic Parkinson's syndrome in vitro, to explore the role of AMPK-mTOR signal pathway in manganese induced autophagy of PC12 cells, and to provide ideas for the pathogenesis and drug research of manganese toxic Parkinson's syndrome. Methods: according to the effect of manganese at different concentration and time on the survival rate of PC12 cells, the cell model of manganese toxic Parkinson's syndrome in vitro was established by selecting appropriate manganese concentration and time of action. The survival rate of PC12 cells was assayed by CCK-8. Ultrastructural changes of PC12 cells treated with manganese were observed by transmission electron microscope. The expression of p-AMPKORp-mTOR pathway key protein p-AMPKORp-4E-BP1 and p-p70S6K in PC12 cells were detected at molecular level. Results the cells were exposed to different manganese concentrations for 24 h, 48 h and 72 h respectively. The survival rate of the cells was measured by CCK-8 method, and the survival rate of PC12 cells was calculated by CCK-8 method. There were significant differences in P0.01between Mn-treated group and control group, P0.01between different groups, and significant difference in P0.01between the same manganese group and control group. The difference was significant according to the control group, Mn-treated group and control group were treated with the control group for 24 h respectively, the CCK-8 method was used to detect and calculate the control group. The cell survival rate of Mn-CC group was significantly lower than that of Control group (P 0.01). Compared with mn CC group, the survival rate of mn CC group was higher than that of mn CC group (P 0.05). The difference was statistically significant. The expression of LC3- 鈪，
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