發(fā)布時(shí)間：2018-06-21 19:14

  本文選題：AMPK + mTOR��； 參考：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:自噬是真核細(xì)胞中降解長壽命蛋白和調(diào)節(jié)細(xì)胞器代謝的主要機(jī)制,受到多種信號(hào)通路的調(diào)控。本研究以錳誘導(dǎo)PC12細(xì)胞建立錳中毒性帕金森綜合征體外細(xì)胞模型,探討AMPK-mTOR信號(hào)通路在錳誘導(dǎo)PC12細(xì)胞自噬中的作用,為錳中毒性帕金森綜合征的發(fā)病機(jī)制及藥物研究提供思路。方法:根據(jù)不同濃度及不同作用時(shí)間錳對(duì)PC12細(xì)胞存活率的影響,選擇合適的錳濃度及作用時(shí)間,建立錳中毒性帕金森綜合征體外細(xì)胞模型,再給予Compound C進(jìn)行干預(yù):CCK-8法檢測(cè)細(xì)胞存活率,透射電子顯微鏡觀察PC12細(xì)胞經(jīng)錳處理后的超微結(jié)構(gòu)變化,分子水平檢測(cè)自噬標(biāo)記蛋白LC3及AMPK-mTOR通路關(guān)鍵蛋白p-AMPK、p-mTOR、p-4E-BP1、p-p70S6K的表達(dá)變化。結(jié)果:1、PC12細(xì)胞在不同錳濃度下分別作用24h、48h、72h,用CCK-8法檢測(cè)計(jì)算細(xì)胞存活率,染錳組與對(duì)照組間比較,P0.01,不同染錳組間比較,P0.01,同一染錳組在不同時(shí)間點(diǎn)間比較,P0.01,差異均具有顯著性。2、PC12細(xì)胞按Control組、Mn組、CC組、Mn+CC組分別加藥處理24h,CCK-8法檢測(cè)計(jì)算Control組細(xì)胞存活率為(100.00±0)%,Mn組、CC組、Mn+CC組細(xì)胞存活率均較Control組下降,P0.01,差異有顯著性,而Mn組與Mn+CC組相比,Mn+CC組細(xì)胞存活率較大,P0.05,差異有統(tǒng)計(jì)學(xué)意義。3、PC12細(xì)胞經(jīng)錳處理后,LC3-Ⅱ/LC3-Ⅰ表達(dá)量增加,呈濃度和時(shí)間依賴性(P㩳0.01)。4、透射電子顯微鏡下可見染錳組細(xì)胞內(nèi)自噬體及自噬溶酶體增加。5、與對(duì)照組相比,Mn組PC12細(xì)胞中p-AMPK、LC3-Ⅱ/LC3-Ⅰ蛋白的相對(duì)表達(dá)量明顯增多(P㩳0.01),p-mTOR、p-p70S6K、p-4E-BP1蛋白的相對(duì)表達(dá)量顯著減少(P㩳0.01);而Mn+CC組中p-AMPK、p-mTOR、p-p70S6K、p-4E-BP1、LC3-Ⅱ/LC3-Ⅰ的表達(dá)量均介于Control組與Mn組之間(P㩳0.05)。結(jié)論:1、錳能誘導(dǎo)PC12細(xì)胞激活自噬,抑制細(xì)胞增殖,且呈濃度和時(shí)間依賴性。2、Compound C可抑制錳對(duì)PC12細(xì)胞自噬的作用。3、錳可能通過AMPK-mTOR信號(hào)通路誘導(dǎo)PC12細(xì)胞激活自噬,抑制細(xì)胞增殖。4、AMPK-mTOR信號(hào)通路可能參與錳中毒性帕金森綜合征的發(fā)病機(jī)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，

本文編號(hào)：2049723