摘要
目的探讨新藤黄酸诱导黑色素瘤B16细胞线粒体自噬的机制。方法 MTT试验检测新藤黄酸对黑色素瘤B16细胞增殖的抑制作用;倒置显微镜观察黑色素瘤B16细胞在新藤黄酸作用下产生的形态变化;透射电镜观察新藤黄酸对黑色素瘤B16细胞线粒体超微结构的影响;流式细胞术检测新藤黄酸诱导黑色素瘤B16细胞线粒体膜电位及ROS的改变;Western blot法检测LC-3、m TOR、AMPK及SIRT3等自噬相关蛋白的变化。结果 MTT结果显示新藤黄酸在体外对黑色素瘤B16细胞的生长及增殖有明显抑制作用,且随着新藤黄酸浓度的增加和作用时间的延长,细胞活力明显下降;倒置显微镜观察黑色素瘤B16细胞显示随着新藤黄酸浓度增加,细胞结构被明显破坏,细胞死亡增多;透射电镜观察发现新藤黄酸作用黑色素瘤B16细胞后,细胞发生明显的线粒体自噬的形态学变化;Western blot法检测表明随着新藤黄酸给药时间的延长,AMPK、SIRT3及LC3-Ⅱ蛋白表达量呈时间依赖性上升,LC3-I蛋白表达量呈时间依赖性下降,m TOR蛋白表达量随着时间延长有所下降。结论新藤黄酸在一定时间和浓度范围内能够抑制黑色素瘤B16细胞的增殖,诱导细胞发生线粒体自噬,其诱导细胞线粒体自噬的机制可能与调控ROS/SIRT3/AMPK信号通路有关。
Objective It is to explore the mechanism of mitoehondrial autophagy in melanoma B16 cell induced by Gambo- genie acid. Methods MTT experiment was used to detect the inhibition of Gambogenie acid on proliferation of melanoma B16 cell. The morphology changes of the cells were observed under inverted microscope. The effect of Gambogenic acid on the mi- toehondrial ultrastructure was observed by transmission electron microscopy. Membrane potential and ROS changes in mito- ehondria were detected by flow eytometry. The correlated protein of mitochondrial autophagy such as LC - 3, mTOR, AMPK and SIRT3 were detected by Western blot method. Results MTT results showed that Gambogenic acid has inhibition on the growth and proliferation of B16 cells, with the increase of concentration and time, the activity of the cells get lower and lower. The results under inverted microscope showed that with the concentration increase, the cell structure was damaged more obvi- ously with more death cells. The results under transmission electron microscopy showed than the obvious morphology changes of B16 ceils like mitochondrial autophagy could be found after induce by Gambogenic acid. Western blot method results showed that the expression of AMPK, SIRT3 and LC3 - 11 had a time dependence increase and LC3 - I had a time depend- ence decrease, that of mTOR decreased a little with time increase. Conclusion Gambogenic acid can inhibit the proliferation of B16 cell in some range of time and dosage, it can induce the mitoehondrial autophagy, and the mechanism may be related with its regulation of ROS/SIRT3/AMPK signal pathway.
出处
《现代中西医结合杂志》
CAS
2016年第19期2079-2082,共4页
Modern Journal of Integrated Traditional Chinese and Western Medicine
