CAJ | 학술논문

目的：探讨紫草素在体内、体外的抗肝癌活性及其可能机制。方法采用Annexin-V FITC/PI双染法检测3种肝癌细胞的细胞凋亡情况，LC3转换法（LC3 Conversion）检测细胞自噬数量，Western blot检测相关凋亡蛋白和自噬标志蛋白的表达，激光共聚焦观察线粒体自噬情况。结果紫草素对3种肝癌细胞均具有杀伤作用，且呈浓度依赖性和时间依赖性，Western blot检测细胞凋亡标志蛋白以及Annexin-V FITC/PI双染检测结果表明，较高浓度紫草素（6～8μM）处理24 h可有效诱导肝癌细胞凋亡。Caspase广谱抑制剂z-VAD预处理细胞可阻止紫草素诱导的肝癌细胞凋亡，紫草素诱导的细胞凋亡依赖于Caspase蛋白的剪切。另一方面，较低浓度紫草素（≤2.5μM）处理肝癌细胞12 h能诱导细胞自噬产生。LC3转换检测、GFP-LC3荧光点聚集和吖啶橙染色自噬泡证明了紫草素能促进细胞自噬的产生；细胞自噬早期抑制剂3-MA能有效阻断紫草素诱导的细胞自噬；LC3周转和p62降解则从动态上证明了自噬潮的产生，激光共聚焦结果紫草素诱导的细胞自噬有线粒体自噬的成分，说明紫草素对线粒体有损伤。结论紫草素能在体外能诱导肝癌细胞发生细胞凋亡和细胞自噬。
목적：탐토자초소재체내、체외적항간암활성급기가능궤제。방법채용Annexin-V FITC/PI쌍염법검측3충간암세포적세포조망정황，LC3전환법（LC3 Conversion）검측세포자서수량，Western blot검측상관조망단백화자서표지단백적표체，격광공취초관찰선립체자서정황。결과자초소대3충간암세포균구유살상작용，차정농도의뢰성화시간의뢰성，Western blot검측세포조망표지단백이급Annexin-V FITC/PI쌍염검측결과표명，교고농도자초소（6～8μM）처리24 h가유효유도간암세포조망。Caspase엄보억제제z-VAD예처리세포가조지자초소유도적간암세포조망，자초소유도적세포조망의뢰우Caspase단백적전절。령일방면，교저농도자초소（≤2.5μM）처리간암세포12 h능유도세포자서산생。LC3전환검측、GFP-LC3형광점취집화아정등염색자서포증명료자초소능촉진세포자서적산생；세포자서조기억제제3-MA능유효조단자초소유도적세포자서；LC3주전화p62강해칙종동태상증명료자서조적산생，격광공취초결과자초소유도적세포자서유선립체자서적성분，설명자초소대선립체유손상。결론자초소능재체외능유도간암세포발생세포조망화세포자서。
Objective To study the activity of shikonin on anti-hepatoma in vitro and in vivo and its possible mechanism.Methods Cell apoptosis was detected by Annexin-V FITC/PI double staining assay;aggregation test was detected by LC3 conversion method in autophagy,Western blot and GFP-LC3 fluorescence point.Results Hepatoma cells and normal liver cells were induced autophagy by shikonin(less than or equal to 2.5 μM)for 12 hours.LC3 conversion,GFP-LC3 fluorescence detection point aggregation and acridine orange staining autophagic vacuoles proved that shikonin induced autophagy.Shikonin induced autophagy could be blocked by early 3-MA autophagy inhibitors effectively;the generation of autophagy tides was proved by LC3 turnover and p62 degradation.Results of apoptosis protein markers detected by Western blot and Annexin-V FITC/PI showed that the higher concentration of shikonin (6 ~8 μM)for 24 hours treatment could induce apoptosis of hepatocellular carcinoma,but couldn't affect the survival of deteriorated liver cells.Apoptosis of hepatocellular carcinoma was prevented by pretreated with broad-spectrum inhibitor z-VAD of Caspase cells.Shikonin induced cell apoptosis depended on the shear of Caspase protein.Conclusion Shikonin could induce the apoptosis and autophagy of hepatocellular carcinoma cells in vitro.