摘要
目的 观察冬凌草甲素（ORI）诱导前列腺癌PC-3细胞自噬,探讨ORI抗前列腺癌的可能机制.方法 实验应用MTT检测细胞存活率、扫描电镜和透射电镜观察细胞超微结构变化、AO染色法观察细胞胞质中酸性泡囊（acidic vesicular organells,AVO）、Western印迹检测MAPI-LC3蛋白水平、RT-PCR技术检测自噬基因beclin 1 mRNA水平.结果 MTT结果显示ORI能有效抑制PC-3细胞增殖,并呈现显著的剂量和时间依赖效应,15 μmol/LORI作用细胞24 h,增殖抑制率达（39.95±3.05）%,48 h后达（51.28±2.91）%.浓度增加至25 μmol/L,抑制率达到（67.94±4.78）%.ORI处理细胞24 h,扫描电镜下观察到细胞缩小变圆,表面微绒毛消失.透射电镜发现细胞核固缩,染色质边集,且胞质中出现大量双层膜包裹形成的自噬体,内含细胞器或胞质.AO染色可见胞质中AVO的形成.Western印迹结果显示ORI促进LC3-Ⅰ向LC3-Ⅱ的转化,ORI处理后LC-Ⅱ蛋白水平升高,LC3-Ⅰ/LC3-Ⅱ比值下降.RT-PCR结果显示自噬抑制剂3-MA能阻止ORI引起的beclin 1 mRNA水平上调.结论 冬凌草甲素能抑制前列腺癌PC-3细胞增殖,并通过上调beclin 1的mRNA水平诱导细胞发生自噬.本课题为冬凌草甲素抗肿瘤机制研究提供了新思路.
Objective To investigate the mechanism of oridonin （ORI）-induced autophagy in prostate cancer PC-3 cells. Methods The PC-3 cells cultured in vitro were treated with ORI. The inhibitory ratio of oridonin In PC-3 cells was assayed by MTT. The ultrastructural cellular changes were observed under light microscope, scanning electron microscope （SEM） and transmission electron microscope （TEM）. AO staining was used to observe the acidic vesicular organelles （AVOs）. The level of MAP1-LC3 was detected by Western blot and reverse transcriptase polymerase chain reaction （RT-PCR） used to detect the level of mRNA of beclin 1. Results After oridonin treatment, the proliferation of PC-3 cells was inhibited significantly in a concentration and time-dependent manner. The SEM examination revealed cellular shrinkage and the disappearance of surface microvilli after ORI treatment. And on the TEM examination, the nuclei exhibited chromatin condensation and the appearance of a large number of autophagosome with doublemembrane structure in cytoplasm. AO staining showed the existence of AVOs. Simultaneously, the expressions of autophagy-related proteins, MAP-LC3 and the mRNA level of beclin 1 were elevated by ORI.The autophagy inhibitor 3-MA reversed the elevation of beclin 1 mRNA. Conclusion The growth of PC-3 cells is inhibited. And cellular is induced by oridonin with a potential anti-tumor effect.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2010年第42期2984-2988,共5页
National Medical Journal of China
基金
基金项目：浙江省中医药科技基金（08029）
