摘要
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。
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 ceils were transfected, and the positive cell clone was selected by using G418. 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 （Sp^mol/1） 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.
出处
《广西医学》
CAS
2013年第8期976-979,983,共5页
Guangxi Medical Journal
基金
广西卫生厅重点科研课题(重2012087)
