CAJ | 학술논문

目的：研究藜芦碱对人肝癌HepG2的细胞毒作用及其诱导细胞凋亡的机制，为开发安全有效的临床用药提供依据。方法藜芦碱0．1～0．6 g·L-1作用于HepG2细胞24 h，CCK-8法检测细胞存活率并计算IC50值；藜芦碱0．1～0．5 g·L-1作用于HepG2细胞24 h，观察细胞形态变化，乳酸脱氢酶（LDH）释放实验检测细胞膜损伤；流式细胞仪检测活性氧（ROS）的生成量、线粒体膜电位的变化以及细胞凋亡的变化；实时荧光定量PCR检测凋亡相关基因p53、Bax、细胞色素c、胱天蛋白酶9和胱天蛋白酶3 mRNA表达。结果藜芦碱0．1～0．6 g·L-1作用于HepG2细胞可以显著抑制细胞活性（P＜0．05，P＜0．01），IC50值为0．4 g·L-1，95％的可信限为0．2558～0．6965 g·L-1。藜芦碱0．1，0．2，0．3，0．4和0．5 g·L-1给药组，与正常对照组相比，LDH释放量显著升高（P＜0．05，P＜0．01），ROS 的生成量显著增大（P＜0．05，P＜0．01），线粒体膜电位显著降低（P＜0．05，P＜0．01），细胞凋亡率显著升高（P＜0．05，P＜0．01）。实时荧光定量PCR检测显示，凋亡相关基因p53、Bax、细胞色素c、胱天蛋白酶9和胱天蛋白酶3 mRNA表达升高（P＜0．05，P＜0．01）。结论藜芦碱具有潜在的细胞毒性，能抑制HepG2细胞增殖，损伤细胞膜和线粒体进而启动凋亡基因胱天蛋白酶9和胱天蛋白酶3 mRNA表达，这可能是其最终导致细胞凋亡的作用机制。
목적：연구려호감대인간암HepG2적세포독작용급기유도세포조망적궤제，위개발안전유효적림상용약제공의거。방법려호감0．1～0．6 g·L-1작용우HepG2세포24 h，CCK-8법검측세포존활솔병계산IC50치；려호감0．1～0．5 g·L-1작용우HepG2세포24 h，관찰세포형태변화，유산탈경매（LDH）석방실험검측세포막손상；류식세포의검측활성양（ROS）적생성량、선립체막전위적변화이급세포조망적변화；실시형광정량PCR검측조망상관기인p53、Bax、세포색소c、광천단백매9화광천단백매3 mRNA표체。결과려호감0．1～0．6 g·L-1작용우HepG2세포가이현저억제세포활성（P＜0．05，P＜0．01），IC50치위0．4 g·L-1，95％적가신한위0．2558～0．6965 g·L-1。려호감0．1，0．2，0．3，0．4화0．5 g·L-1급약조，여정상대조조상비，LDH석방량현저승고（P＜0．05，P＜0．01），ROS 적생성량현저증대（P＜0．05，P＜0．01），선립체막전위현저강저（P＜0．05，P＜0．01），세포조망솔현저승고（P＜0．05，P＜0．01）。실시형광정량PCR검측현시，조망상관기인p53、Bax、세포색소c、광천단백매9화광천단백매3 mRNA표체승고（P＜0．05，P＜0．01）。결론려호감구유잠재적세포독성，능억제HepG2세포증식，손상세포막화선립체진이계동조망기인광천단백매9화광천단백매3 mRNA표체，저가능시기최종도치세포조망적작용궤제。
OBJECTIVE Tostudythehepatotoxicityofveratrinehydrochloride(VH)anditsmecha-nismoninductionofapoptosisinvitro.METHODS HepG2cellswereexposedtoVH0.1-0.6g·L-1 for 24 h,cell viability was examined by CCK-8 assay,and the morphologic changes in HepG2 cells were quantified.After the treatment with VH 0.1 -0.5 g·L-1 for 24 h,cell membrane injury was examined by detecting the release rate of lactate dehydrogenase (LDH).The effect on reactive oxygen species (ROS),mitochondrial membrane potential and apoptosis was detected by flow cytometry.The mRNA expression of p53,Bax,cytochrome c,caspase 9,caspase 3 was evaluated by real-time PCR. RESULTS HepG2cellviabilitywassignificantlyreducedfollowingexposuretoVH0.1-0.5g·L-1. The IC50 value was 0.4 g·L-1 .The 95%confidence limit was 0.2558-0.6965 g·L-1 .The LDH release rate,ROS and apoptosis rate of HepG2 cells were significantly increased after exposure to VH 0.1 -0.5 g·L-1 for 24 h (P<0.05,P<0.01 ),and the mitochondrial membrane potential markedly declined (P<0.05,P<0.01 ).The expression of p53,Bax,cytochrome c,caspase 9 and caspase 3 was increased(P<0.05,P<0.01).CONCLUSION VHhascytotoxicpotential.Damagetocell me mbrane and mitochondria and initiation of apoptosis-related genes of caspase 9 and caspase 3 mRNA expression may be the mechanis m of apoptosis.