摘要
目的研究Aβ25-35对PC12细胞内Ca2+浓度和自噬发生的影响。方法培养PC12细胞,分别用0、5、10μmol/L Aβ25-35处理24 h,用MTT法测定细胞存活率,流式细胞仪检测细胞内Ca2+浓度,单丹磺酰戊二胺(monodansylcadaverine,MDC)染色观察自噬泡,共聚焦显微镜检测LC3-Ⅱ表达,透射电镜观察细胞超微结构。结果 5、10μmol/L Aβ25-35处理24 h后,PC12细胞存活率分别为(64.6±2.3)%和(41.3±4.1)%,与对照组相比显著下降(P<0.05),细胞内游离Ca2+浓度升高(P<0.05)。与此同时,随着Aβ25-35浓度升高,显微镜下可见细胞皱缩、变圆,与对照组相比,细胞内MDC阳性颗粒增多,LC3-Ⅱ荧光强度增强(P<0.05),与Aβ25-35呈浓度依赖关系。电镜下可见单(双)层膜样自噬体。结论Aβ25-35诱导PC12细胞发生自噬呈浓度依赖关系,其发生可能与细胞内游离Ca2+浓度升高有关。
Objective To study the effects of Aβ25-35 on intracellular Ca2＋ concentration and autophagy in PC12 cells.Methods PC12 cells were treated with 0, 5, and 10 μmol/L Aβ25-35 for 24 h. Cell viability was detected by MTT assay. The intracellular Ca2＋ concentration was measured by flow cytometry. The autophagic vacuoles were observed by monodansylcadaverine （MDC） fluorescent staining. The expression of LC3-Ⅱ was examined by fluorescent staining. The ultrastructure of cells were observed by transmission electronic microscopy.Results The viability of PC12 cells after incubation with 5 and 10 μmol/L Aβ25-35 was （64.6±2.3）% and （41.3±4.1）%, respectively, which were significantly decreased as compared to the control group （P〈0.05）. The intracellular Ca2＋ concentrations of the PC12 cells after incubation with 5 and 10 μmol/L Aβ25-35 were significantly higher than that of the control group （P〈0.05）. Meanwhile, it could be detected that the cells after Aβ25-35 treatment became shrink and round. The autophagic vacuoles stained with MDC in Aβ25-35-treated groups were significantly more than the in control group （P〈0.05）. The expression level of LC3-Ⅱ was elevated after treated with Aβ25-35 （P〈0.05） in a dose-dependent manner. Autophagosomes were observed after Aβ25-35 treatment.Conclusion Aβ25-35 may induce autophagy in PC12 cells in a dose-dependent manner. The mechanism might be related to the increase of intracellular Ca2＋ concentration.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2014年第4期355-359,共5页
Journal of Third Military Medical University
基金
国家自然科学基金(30470608
30500171)~~
