摘要
目的对丙戊酸诱导胶质瘤细胞发生自噬性死亡的过程进行观察,并初步探讨其可能的机制。方法丙戊酸处理人脑胶质瘤细胞系U87、T98G和SF295,台盼蓝染色检测细胞活性并计算细胞存活率;流式细胞仪测定细胞周期,透射电子显微镜和荧光测定仪观察细胞超微结构及自噬水平,West-ern blotting检测经丙戊酸处理后细胞内LC3-Ⅱ、Beclin-1、p-Akt和p-p70S6K等蛋白质的表达水平。结果经不同浓度丙戊酸处理后,人脑胶质瘤细胞系U87、T98G和SF295细胞活性明显受到抑制,其胶质瘤细胞半数抑制浓度分别为(2.45±0.32)、(4.78±0.62)和(6.62±0.95)mmol/L,其中丙戊酸对人脑胶质瘤细胞系U87具有较明显的杀伤作用(P<0.05)。透射电子显微镜观察可见人脑胶质瘤细胞系U87细胞胞质内有大量自噬体和自噬溶酶体,其自噬水平随丙戊酸浓度的升高而逐渐增强。经丙戊酸处理后,人脑胶质瘤细胞系U87细胞自噬相关蛋白LC3-Ⅱ和Beclin-1表达水平明显升高,而p-Akt和p-p70S6K表达水平明显降低。结论丙戊酸可诱导胶质瘤细胞发生自噬,其诱导胶质瘤细胞发生自噬的机制可能与阻断Akt信号转导通路有关。
Objective To-observe the autophagy of glioma cell induced by valproic acid (VPA) and explore the possible mechanism. Methods Human cerebral glioma cell line U87, T98G and SF295 treated with VPA were used in this study. Cell viability was determined by trypan blue staining. Cell cycle was analysed by flow cytometry after PI staining. Autophagosome was observed under transmission electronic microscopy. The intensity of autophagy was analysed by monodansylcadaverine (MDC) fluorescent staining. The expression levels of LC3- Ⅱ, Beclin-1, p-Akt and p-p70S6K were measured by Western blotting and optical densitometry analysis after VPA treatment. Statistical analysis was performed by using student t-test (two tailed). The criterion for statistical significance was taken as P ≤ 0.05. Results The viability of human cerebral glioma cell line U87, T98G and SF295 were obviously inhibited after incubation with different concentrations of VPA, half of inhibition concentration (IC50) values were (2.45 ± 0.32), (4.78 ± 0.62) and (6.62 ± 0.95) mmol/L, respectively. VPA exhibited better cytotoxicity effect on human cerebral glioma cell line U87 (P〈 0.05). Autophagosomes and autolysosomes were observed in VPA-treated human cerebral glioma ceil line U87 under transmission electronic microscopy. The intensity of autopbagy was correlated with concentrations of VPA in glioma cell. The expression levels of related protein LC3- Ⅱ and Beclin-1 were elevated, while p- Akt and p-p70S6K were declined following VPA treatment in human cerebral glioma cell line U87. Conclusion VPA may induce autophagy in glioma cell. The mechanism may be related to the disruption of Akt signaling pathway.
出处
《中国现代神经疾病杂志》
CAS
2008年第5期460-464,共5页
Chinese Journal of Contemporary Neurology and Neurosurgery
基金
国家自然科学基金资助项目(项目编号:30772551)
华南肿瘤学国家重点实验室985-Ⅱ基金资助项目