【摘要】：目的：探討皮質(zhì)酮對PC12細(xì)胞的損傷是否通過自噬溶酶體途徑。 方法： 1. PC12細(xì)胞進(jìn)行隨機實驗分組：皮質(zhì)酮組（設(shè)三個劑量組：1×10-7M；1×10-6M和1×10-5M）和溶媒（DMSO）對照組，分別孵育24小時后，采用MTT法檢測細(xì)胞活力，流式細(xì)胞術(shù)檢測細(xì)胞凋亡，免疫印跡方法檢測自噬溶酶體標(biāo)記蛋白LC3-II/LC3-I、p62及凋亡相關(guān)蛋白Caspase-3的表達(dá)，腺病毒熒光雙標(biāo)載體轉(zhuǎn)染PC12細(xì)胞，激光共聚焦觀察自噬流的變化。探討糖皮質(zhì)激素對PC12細(xì)胞損傷與自噬溶酶體途徑的影響。 2. PC12細(xì)胞實驗分組：（1）皮質(zhì)酮（1×10-5M）組（2）皮質(zhì)酮+氯喹（10μM）組（氯喹組）（3）皮質(zhì)酮+雷帕霉素（20μM）組（雷帕霉素組）（4）溶媒（DMSO）對照組，分別孵育24小時后，以流式細(xì)胞術(shù)檢測細(xì)胞凋亡，免疫印跡方法檢測自噬溶酶體標(biāo)記蛋白LC3-II/LC3-I、p62及凋亡相關(guān)蛋白Caspase-3的表達(dá)，腺病毒熒光雙標(biāo)載體轉(zhuǎn)染PC12細(xì)胞，激光共聚焦觀察自噬流的變化，闡明自噬溶酶體途徑在皮質(zhì)酮誘導(dǎo)細(xì)胞損傷中的作用機制。 結(jié)果： 1. MTT法檢測結(jié)果表明，高濃度皮質(zhì)酮顯著促進(jìn)PC12細(xì)胞的損傷；流式細(xì)胞術(shù)檢測結(jié)果顯示，高濃度皮質(zhì)酮明顯增加細(xì)胞的凋亡；免疫印跡技術(shù)結(jié)果顯示，高濃度皮質(zhì)酮顯著上調(diào)PC12細(xì)胞LC3-II、p62及凋亡相關(guān)蛋白Caspase-3的表達(dá)，升高LC3-II/LC3-I的比值；激光共聚焦檢測結(jié)果表明，，高濃度皮質(zhì)酮顯著增加PC12細(xì)胞自噬體的數(shù)量，而自噬溶酶體的數(shù)量相對減少。 2.自噬溶酶體途徑抑制劑顯著增加高濃度皮質(zhì)酮組PC12細(xì)胞的損傷與凋亡，上調(diào)皮質(zhì)酮組PC12細(xì)胞LC3-II、p62及Caspase-3的表達(dá)、升高LC3-II/LC3-I的比值，該組細(xì)胞的自噬體數(shù)量明顯增加、而自噬溶酶體數(shù)量顯著減少。自噬溶酶體途徑誘導(dǎo)劑雷帕霉素則能逆轉(zhuǎn)高濃度皮質(zhì)酮的上述作用。 結(jié)論： 皮質(zhì)酮能促進(jìn)PC12細(xì)胞的損傷與凋亡，并誘導(dǎo)自噬溶酶體途徑功能障礙。皮質(zhì)酮損傷PC12細(xì)胞可能通過自噬溶酶體途徑依賴機制。
[Abstract]:Aim: to investigate whether corticosterone can damage PC12 cells through autophagy lysosome pathway. Methods: 1. PC12 cells were randomly divided into two groups: corticosterone group (3 dose groups: 1 脳 10-7m; The control group (1 脳 10-6m and 1 脳 10-5m) and the solvent (DMSO) control group were incubated for 24 hours. The cell viability was detected by MTT method, the apoptosis was detected by flow cytometry, and the autophagy lysosome labeled protein LC3-II/LC3-I, was detected by immunoblotting. The expression of p62 and apoptosis-related protein Caspase-3 was detected by confocal laser scanning. The expression of p62 and apoptosis-related protein Caspase-3 was detected by confocal laser scanning. To investigate the effects of glucocorticoid on PC12 cell injury and autophagy lysosome pathway. 2. PC12 cells were divided into two groups: (1) corticosterone (1 脳 10-5m) group (2) corticosterone chloroquine (10 渭 M) group (chloroquine group) (3) corticosterone rapamycin (20 渭 M) group (rapamycin group) (4) solvent (DMSO) control group. After 24 hours incubation, apoptosis was detected by flow cytometry, the expression of autophagy lysosome-labeled protein LC3-II/LC3-I,p62 and apoptosis-related protein Caspase-3 was detected by immunoblotting, and adenovirus double-labeled vector was transfected into PC12 cells. The mechanism of autophagy lysosome pathway in corticosterone-induced cell injury was elucidated by confocal laser scanning. Results: 1. The results of MTT assay showed that high concentration of corticosterone significantly promoted the injury of PC12 cells, and the results of flow cytometry showed that high concentration of corticosterone significantly increased the apoptosis of PC12 cells. Western blot showed that high concentration of corticosterone could significantly up-regulate the expression of LC3-II,p62 and apoptosis-related protein Caspase-3 and increase the ratio of LC3-II/LC3-I in PC12 cells. Confocal laser scanning showed that high concentration of corticosterone significantly increased the number of autophagy in PC12 cells, but decreased the number of autophagy lysosomes. 2. Autophagy lysosome pathway inhibitor significantly increased the injury and apoptosis of PC12 cells in high concentration corticosterone group, up-regulated the expression of LC3-II,p62 and Caspase-3 in PC12 cells and increased the ratio of LC3-II/LC3-I in corticosterone group. The number of autophagy increased significantly, but the number of autophagy lysosome decreased significantly. Rapamycin, an inducer of autophagy lysosome pathway, could reverse the above effects of high concentration corticosterone. Conclusion: corticosterone can promote the injury and apoptosis of PC12 cells and induce autophagy lysosome pathway dysfunction. Corticosterone-induced injury of PC12 cells may be mediated by autophagy lysosome pathway.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

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