皮质酮诱导PC12细胞损伤的自噬—溶酶体途径依赖机制
发布时间：2019-04-28 07:01
【摘要】：目的：探讨皮质酮对PC12细胞的损伤是否通过自噬溶酶体途径。 方法： 1. PC12细胞进行随机实验分组：皮质酮组（设三个剂量组：1×10-7M；1×10-6M和1×10-5M）和溶媒（DMSO）对照组，分别孵育24小时后，采用MTT法检测细胞活力，流式细胞术检测细胞凋亡，免疫印迹方法检测自噬溶酶体标记蛋白LC3-II/LC3-I、p62及凋亡相关蛋白Caspase-3的表达，腺病毒荧光双标载体转染PC12细胞，激光共聚焦观察自噬流的变化。探讨糖皮质激素对PC12细胞损伤与自噬溶酶体途径的影响。 2. PC12细胞实验分组：（1）皮质酮（1×10-5M）组（2）皮质酮+氯喹（10μM）组（氯喹组）（3）皮质酮+雷帕霉素（20μM）组（雷帕霉素组）（4）溶媒（DMSO）对照组，分别孵育24小时后，以流式细胞术检测细胞凋亡，免疫印迹方法检测自噬溶酶体标记蛋白LC3-II/LC3-I、p62及凋亡相关蛋白Caspase-3的表达，腺病毒荧光双标载体转染PC12细胞，激光共聚焦观察自噬流的变化，阐明自噬溶酶体途径在皮质酮诱导细胞损伤中的作用机制。 结果： 1. MTT法检测结果表明，高浓度皮质酮显著促进PC12细胞的损伤；流式细胞术检测结果显示，高浓度皮质酮明显增加细胞的凋亡；免疫印迹技术结果显示，高浓度皮质酮显著上调PC12细胞LC3-II、p62及凋亡相关蛋白Caspase-3的表达，升高LC3-II/LC3-I的比值；激光共聚焦检测结果表明，，高浓度皮质酮显著增加PC12细胞自噬体的数量，而自噬溶酶体的数量相对减少。 2.自噬溶酶体途径抑制剂显著增加高浓度皮质酮组PC12细胞的损伤与凋亡，上调皮质酮组PC12细胞LC3-II、p62及Caspase-3的表达、升高LC3-II/LC3-I的比值，该组细胞的自噬体数量明显增加、而自噬溶酶体数量显著减少。自噬溶酶体途径诱导剂雷帕霉素则能逆转高浓度皮质酮的上述作用。 结论： 皮质酮能促进PC12细胞的损伤与凋亡，并诱导自噬溶酶体途径功能障碍。皮质酮损伤PC12细胞可能通过自噬溶酶体途径依赖机制。
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
本文编号：2467418
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
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