CAJ | 학술논문

目的：研究香加皮杠柳苷(CPP)对人肺癌 A549细胞凋亡及 survivin 表达的影响,探讨其抗肿瘤作用及作用机制。方法采用噻唑蓝(MTT)法检测1.25,2.50,5.00,10.00,20.00 ng·mL-1 CPP 处理24,48,72 h 对人肺癌 A549细胞的抑制作用；应用流式细胞术分析不同浓度 CPP(2.50,5.00,10.00 ng·mL-1)分别作用于 A549细胞6,12,24,48,72 h 对细胞凋亡和凋亡率的影响；应用吖啶橙/溴乙啶(AO/ EB)荧光染色法和透射电镜观察 CPP 处理前后 A549细胞凋亡的形态学及超微结构变化；采用反转录聚合酶链反应(RT-PCR)法检测 CPP 作用后 A549细胞中凋亡抑制基因 survivin的 mRNA 表达情况；应用 Western blot 检测 CPP 对 A549细胞 survivin 蛋白表达的影响。结果 CPP 能显著抑制人肺癌A549细胞的生长,最大抑制率(93.46±2.35)%。流式细胞术结果显示,CPP 处理组可见典型的凋亡峰,与对照组比较, A549细胞的凋亡率明显升高(P<0.05)。荧光显微镜下可见 CPP 处理组 A549细胞呈橘红色的凋亡细胞形态。透射电镜下可见经 CPP 处理的 A549细胞体积变小,出现细胞质凝缩,核内染色质凝聚边集于核膜,内质网扩张,细胞质空泡化等凋亡细胞的特征性超微结构改变。 RT-PCR 结果显示,经 CPP 处理的 A549细胞中 survivin mRNA 表达降低(P<0.05),10.0 ng·mL-1 CPP 组 survivin mRNA 表达由对照组的(0.928±0.016)降至(0.251±0.012)；Western blot 结果显示 CPP 组细胞中 survivin 蛋白表达明显减弱。结论 CPP 可诱导人肺癌 A549细胞发生凋亡,可通过下调 survivin 基因的 mRNA和蛋白表达而发挥诱导细胞凋亡作用。
목적：연구향가피강류감(CPP)대인폐암 A549세포조망급 survivin 표체적영향,탐토기항종류작용급작용궤제。방법채용새서람(MTT)법검측1.25,2.50,5.00,10.00,20.00 ng·mL-1 CPP 처리24,48,72 h 대인폐암 A549세포적억제작용；응용류식세포술분석불동농도 CPP(2.50,5.00,10.00 ng·mL-1)분별작용우 A549세포6,12,24,48,72 h 대세포조망화조망솔적영향；응용아정등/추을정(AO/ EB)형광염색법화투사전경관찰 CPP 처리전후 A549세포조망적형태학급초미결구변화；채용반전록취합매련반응(RT-PCR)법검측 CPP 작용후 A549세포중조망억제기인 survivin적 mRNA 표체정황；응용 Western blot 검측 CPP 대 A549세포 survivin 단백표체적영향。결과 CPP 능현저억제인폐암A549세포적생장,최대억제솔(93.46±2.35)%。류식세포술결과현시,CPP 처리조가견전형적조망봉,여대조조비교, A549세포적조망솔명현승고(P<0.05)。형광현미경하가견 CPP 처리조 A549세포정귤홍색적조망세포형태。투사전경하가견경 CPP 처리적 A549세포체적변소,출현세포질응축,핵내염색질응취변집우핵막,내질망확장,세포질공포화등조망세포적특정성초미결구개변。 RT-PCR 결과현시,경 CPP 처리적 A549세포중 survivin mRNA 표체강저(P<0.05),10.0 ng·mL-1 CPP 조 survivin mRNA 표체유대조조적(0.928±0.016)강지(0.251±0.012)；Western blot 결과현시 CPP 조세포중 survivin 단백표체명현감약。결론 CPP 가유도인폐암 A549세포발생조망,가통과하조 survivin 기인적 mRNA화단백표체이발휘유도세포조망작용。
Objective To investigate the effects of periplocin from Cortex Periplocae (CPP) on apoptosis of human lung cancer A549 cells and expression of survivin, and demonstrate its anti-tumor effect and the possible mechanism. Methods Inhibitory effect of CPP at different concentrations (1. 25, 2. 50, 5. 00, 10. 00, 20. 00 ng·mL-1 ) and for different time length (24, 48, 72 h) on A549 cell proliferation was tested by MTT method. Apoptosis rate of A549 cells treated with CPP at different concentrations (2. 50, 5. 00, 10. 00 ng·mL-1 ) were measured using flow cytometry (FCM) for 6, 12, 24, 48, 72 h, respectively. The morphological and ultrastructural changes of the apoptosis cells were observed by acridine orange/ ethidium bromide (AO/ EB) staining and transmission electron microscopy (TEM). The effects of CPP on mRNA and protein expression of apoptosis associated gene survivin were assessed by RT-PCR and Western blotting. Results CPP could significantly inhibit the growth of A549, and the inhibition rate reached (93. 46±2. 35)% . The results of FCM showed that the apoptosis rate of A549 cells treated with CPP was increased significantly as compared to the control group ( P<0. 05). Meanwhile, typical apoptotic peaks were detected. The characteristic morphological changes of apoptosis were observed in A549 exposed to CPP, including cell shrinkage, the nuclei became yellow-red by AO/ EB staining, and typical ultrastructural changes, including nuclear chromatin condensation along the nuclear membrane, vacuolar degeneration of cytoplasm observed by TEM. The result of RT-PCR indicated that survivin mRNA expression decreased obviously in A549 cells exposed to CPP. The protein expression of survivin in A549 cells treated with 10. 0 ng·mL-1 CPP(0. 251±0. 012)was weaker than that in control group(0. 928±0. 016). Conclusion CPP can induce apoptosis in human lung cancer cell lines A549, and the probable mechanism is related to the down-regulation of survivin mRNA and protein.