CAJ | 학술논문

目的探讨乙肝病毒HBx蛋白对索拉非尼诱导的肝癌细胞凋亡的影响。方法将空载质粒pEGFP-N1、目的质粒pEGFP-N1-HBx扩增，转染HepG 2细胞，G418筛选抗性克隆，并用RT-PCR检测细胞中HBx表达以表明建立稳定表达细胞株HepG 2/GFP-HBx。用索拉非尼（5μmol/L）分别处理HepG 2、HepG 2/GFP、HepG 2/GFP-HBx细胞，CCK-8计算不同时间点的抑制率；流式细胞检测处理48 h后凋亡率。结果 pEG-FP-N1-HBx经PCR验证扩增成功，RT-PCR显示HepG 2/GFP-HBx细胞有HBx基因转录表达。 CCK-8表明索拉非尼处理的3组细胞均发生了明显的时间依赖性细胞凋亡，流式细胞检测显示索拉非尼处理48 h后，HepG 2、HepG 2/GFP细胞凋亡率分别为32．76％、32．80％明显高于HepG 2/GFP-HBx（16．92％）（P＜0．01）；未处理组细胞凋亡率为3．72％、4．40％、3．85％，组间比较差异无统计学意义（P＞0．05）。结论成功建立了稳定表达GFP、GFP-HBx的HepG 2细胞模型；HBx能够抑制索拉非尼诱导的HepG 2细胞凋亡。
목적탐토을간병독HBx단백대색랍비니유도적간암세포조망적영향。방법장공재질립pEGFP-N1、목적질립pEGFP-N1-HBx확증，전염HepG 2세포，G418사선항성극륭，병용RT-PCR검측세포중HBx표체이표명건립은정표체세포주HepG 2/GFP-HBx。용색랍비니（5μmol/L）분별처리HepG 2、HepG 2/GFP、HepG 2/GFP-HBx세포，CCK-8계산불동시간점적억제솔；류식세포검측처리48 h후조망솔。결과 pEG-FP-N1-HBx경PCR험증확증성공，RT-PCR현시HepG 2/GFP-HBx세포유HBx기인전록표체。 CCK-8표명색랍비니처리적3조세포균발생료명현적시간의뢰성세포조망，류식세포검측현시색랍비니처리48 h후，HepG 2、HepG 2/GFP세포조망솔분별위32．76％、32．80％명현고우HepG 2/GFP-HBx（16．92％）（P＜0．01）；미처리조세포조망솔위3．72％、4．40％、3．85％，조간비교차이무통계학의의（P＞0．05）。결론성공건립료은정표체GFP、GFP-HBx적HepG 2세포모형；HBx능구억제색랍비니유도적HepG 2세포조망。
Objective To investigate the effect of hepatitis B virus X ( HBx) protein on sorafenib-induced ap-optosis of hepatocellar carcinoma cells .Methods The pEGFP-N1 and pEGFP-N1-HBx were amplified ,HepG2 cells were transfected ,and the positive cell clone was selected by using G 418 .Reverse transcription polymerase chain reac-tion(RT-PCR) was used to detect the expression of HBx gene for demonstration of stable expression of cell line HepG2/GFP-HBx.HepG2,HepG2/GFP and HepG2/GFP-HBx cells were treated with sorafenib (5μmol/l) respec-tively ,the inhibition rates of the cells at different time points were tested by CCK-8;Flow cytometry was used to test the apoptosis rates of the cells 48 hours after incubation .Results The recombinant plasmid pEGFP-N1-HBx was ver-ified to contain HBx by PCR amplification .The HBx expression was detected by RT-PCR.CCK-8 showed that sor-afenib induced significant time-dependent cell apoptosis in HepG2,HepG2/GFP and HepG2/GFP-HBx cells.Flow cytometry showed that the apoptosis rates in HepG2(32.76%),HepG2/GFP(32.80%) cells were significantly high-er than that in HepG2/GFP-HBx cell(16.92%) 48 hours after incubation of sorafenib (P<0.01); The apoptosis rates in untreated cells were 3.72%,4.40%,3.85%,respectively,there was no significant difference among groups (P>0.05).Conclusion The stable-expression HepG2/GFP and HepG2/GFP-HBx cell models are established suc-cessfully;HBx protein can suppress sorafenib-induced apoptosis of HepG2 cells.