CAJ | 학술논문

目的观察索拉非尼联合奥曲肽在体外对人肝癌细胞株HepG2的抑制作用,并探讨其机制. 方法采用不同浓度梯度的索拉非尼、奥曲肽单独和联合作用于人肝癌细胞株HepG2,分别于24、48、72 h用CCK-8试剂盒测定各组细胞的杀伤效应,流式细胞术检测HepG2细胞的凋亡率,荧光显微镜观察细胞的凋亡情况；并用酶联免疫吸附法检测细胞培养上清液中血管内皮生长因子(VEGF)的水平,Western blot法测定细胞中髓样细胞白血病-1(Mcl-1)、细胞外信号调节激酶(ERK) 1/2、磷酸化ERK1/2的表达.数据采用均数±标准差((x)±s)表示,用SPSS17.0软件对数据进行单因素方差分析. 结果索拉非尼、奥曲肽单独使用和联合使用均对HepG2细胞具有抑制作用,并诱导其凋亡,联合用药组抑制效果优于单独用药组F=200.398,(P＜0.05).荧光显微镜观察显示,联合用药组和单独用药组细胞膜上均出现代表细胞早期凋亡的标志物磷脂酰丝氨酸.联合用药组VEGF表达水平为(10.31±4.69) pg/ml,低于索拉非尼组[(22.73±5.88)pg/ml]、奥曲肽组[(25.46±3.45) pg/ml]及正常对照组[(57.15±6.32) pg/ml],F=1019.725,P值均＜0.05.Western blot 法结果显示,各组ERK1/2表达水平差异并无统计学意义(P＞0.05)；与正常对照相组比,索拉非尼、奥曲肽及联合用药组的磷酸化ERK1/2表达均减少(F=2.401,P＜0.05),联合用药组表达水平低于单独用药组(P值均＜0.05)；奥曲肽组Mcl-1的表达水平与对照组差异无统计学意义,索拉非尼组和联合用药组的表达水平低于正常对照组(P值均＜ 0.05),联合用药组的Mcl-1表达水平低于各单独用药组(P值均＜ 0.05).结论索拉非尼、奥曲肽均可抑制人肝癌细胞HepG2增殖,并诱导其凋亡,联合应用效果更为显著；其机制可能与二者协同抑制VEGF的表达,并下调抗凋亡蛋白Mcl-1及磷酸化ERK1/2的表达有关.
목적 관찰색랍비니연합오곡태재체외대인간암세포주HepG2적억제작용,병탐토기궤제. 방법 채용불동농도제도적색랍비니、오곡태단독화연합작용우인간암세포주HepG2,분별우24、48、72 h용CCK-8시제합측정각조세포적살상효응,류식세포술검측HepG2세포적조망솔,형광현미경관찰세포적조망정황；병용매련면역흡부법검측세포배양상청액중혈관내피생장인자(VEGF)적수평,Western blot법측정세포중수양세포백혈병-1(Mcl-1)、세포외신호조절격매(ERK) 1/2、린산화ERK1/2적표체.수거채용균수±표준차((x)±s)표시,용SPSS17.0연건대수거진행단인소방차분석. 결과 색랍비니、오곡태단독사용화연합사용균대HepG2세포구유억제작용,병유도기조망,연합용약조억제효과우우단독용약조F=200.398,(P＜0.05).형광현미경관찰현시,연합용약조화단독용약조세포막상균출현대표세포조기조망적표지물린지선사안산.연합용약조VEGF표체수평위(10.31±4.69) pg/ml,저우색랍비니조[(22.73±5.88)pg/ml]、오곡태조[(25.46±3.45) pg/ml]급정상대조조[(57.15±6.32) pg/ml],F=1019.725,P치균＜0.05.Western blot 법결과현시,각조ERK1/2표체수평차이병무통계학의의(P＞0.05)；여정상대조상조비,색랍비니、오곡태급연합용약조적린산화ERK1/2표체균감소(F=2.401,P＜0.05),연합용약조표체수평저우단독용약조(P치균＜0.05)；오곡태조Mcl-1적표체수평여대조조차이무통계학의의,색랍비니조화연합용약조적표체수평저우정상대조조(P치균＜ 0.05),연합용약조적Mcl-1표체수평저우각단독용약조(P치균＜ 0.05).결론 색랍비니、오곡태균가억제인간암세포HepG2증식,병유도기조망,연합응용효과경위현저；기궤제가능여이자협동억제VEGF적표체,병하조항조망단백Mcl-1급린산화ERK1/2적표체유관.
Objective To investigate the effects of sorafenib and octreotide combination treatment on cellular proliferation and explore the underlying molecular mechanisms by using an in vitro cell culture system with the human hepatoma cell line,HepG2.Methods HepG2 cells were treated with different concentrations of sorafenib and octreotide alone or in combination.Untreated HepG2 cells were used as controls.Treatment-induced cytotoxicity was determined with the cell counting kit-8 by Sigma-Aldrich,and rate of apoptosis was detected by flow cytometry.Fluorescent microscopy was used to observe rates of cell growth under the various treatments.Treatment-induced changes in protein expressions were detected by enzyme-linked immunosorbent assay (for vascular endothelial growth factor (VEGF)) and Western blotting (for the Mcl-1 apoptosis mediator and the ERK1/2 and PERK1/2 kinases).Result Sorafenib and octreotide,used alone or in combination,inhibited proliferation and induced apoptosis in HepG2 cells.Combination treatment was more effective than either mono-treatment (F =200.398,P ＜ 0.05).Fluorescent microscopy showed that combination treatment stimulated phosphatidylserine,the marker of early apoptosis,better than either mono-treatment.VEGF expression in cultures exposed to combination treatment was also significantly lower than in mono-treatment or untreated control cultures (F =1019.725,P ＜ 0.05).Western blotting showed that octreotide mono-treatment had no effect on Mcl-1 expression (vs.control group; P ＞ 0.05) and that combination treatment significantly lowered Mcl-1 expression (vs.mono-treatment and control groups; P ＜0.05).None of the treatments affected ERK1/2 expression (all,P ＞ 0.05),while all treatments significantly lowered PERK1/2 expression (vs.control group; F =2.401,P ＜ 0.05) and the combination treatment lowered PERK1/2 significantly more than either mono-treatment (P ＜ 0.05).Conclusions Sorafenib and octreotide can inhibit proliferation and induce apoptosis in the human hepatoma cell line,HepG2.Combination treatment is significantly more efficacious (P ＜ 0.05) and produced synergistic effects.The mechanism underlying this phenomenon may depend on synergistic inhibition of VEGF,the anti-apoptotic protein Mcl-1,and the proliferation-inducing PERK1/2.