CAJ | 학술논문

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.
목적 관찰동릉초갑소(ORI)유도전렬선암PC-3세포자서,탐토ORI항전렬선암적가능궤제.방법 실험응용MTT검측세포존활솔、소묘전경화투사전경관찰세포초미결구변화、AO염색법관찰세포포질중산성포낭(acidic vesicular organells,AVO)、Western인적검측MAPI-LC3단백수평、RT-PCR기술검측자서기인beclin 1 mRNA수평.결과 MTT결과현시ORI능유효억제PC-3세포증식,병정현현저적제량화시간의뢰효응,15 μmol/LORI작용세포24 h,증식억제솔체(39.95±3.05)%,48 h후체(51.28±2.91)%.농도증가지25 μmol/L,억제솔체도(67.94±4.78)%.ORI처리세포24 h,소묘전경하관찰도세포축소변원,표면미융모소실.투사전경발현세포핵고축,염색질변집,차포질중출현대량쌍층막포과형성적자서체,내함세포기혹포질.AO염색가견포질중AVO적형성.Western인적결과현시ORI촉진LC3-Ⅰ향LC3-Ⅱ적전화,ORI처리후LC-Ⅱ단백수평승고,LC3-Ⅰ/LC3-Ⅱ비치하강.RT-PCR결과현시자서억제제3-MA능조지ORI인기적beclin 1 mRNA수평상조.결론 동릉초갑소능억제전렬선암PC-3세포증식,병통과상조beclin 1적mRNA수평유도세포발생자서.본과제위동릉초갑소항종류궤제연구제공료신사로.
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.