转基因细胞模型L02/HBx的构建及HBx对细胞周期的影响

Establishment of genetransfected cell strain LO2/HBx and effect of HBx on the cell cycles

目的:构建L02/HBx转基因细胞模型并研究HBx对肝细胞周期的影响.方法:运用脂质体转染和G418筛选获得L02/ HBx阳性克隆,并分别用RT-PCR和Western blot鉴定HBx mRNA与蛋白的表达.进一步用四唑蓝(MTT)比色试验、流式细胞仪检测L02/HBx的增殖、凋亡和细胞周期.结果:RT-PCR和Western blot分别检测到L02/ HBx细胞中HBx mRNA和蛋白的表达.MTT比色试验显示L02/HBx生长速度加快,流式细胞仪检测发现L02/HBx凋亡率低(0.09%±0.13% vs 3.74%±1.29%,P<0.05),G1期细胞比例减少(61.35%±0.82% vs 67.80±6.84%,P<0.05),S期细胞比例相应增加(36.59%±2.54% vs 22.37%±2.17%,P<0.05).经阿霉素(ADM)培养后,L02/HBx的凋亡率显著增加(34.91%±5.85% vs 0.09%±0.13%,P<0.05),G1期细胞比例明显增加但低于对照组(82.81%±6.48% vs 61.35%±0.82%,P<0.05;82.81%±6.48% vs 87.19%±1.92%,P<0.05),S期细胞比例降低但较对照组高(13.84%±6.16% vs 36.59%±2.54%,P<0.05;13.84%±6.16% vs 2.22%±1.26%,P<0.05).结论:L02/HBx构建成功,HBx能促进细胞周期进程,加快细胞的生长并抑制细胞的凋亡;转染HBx基因的肝细胞凋亡更易受凋亡因子所触发,表明HBx可能会增加正常肝细胞对诱导凋亡因素的敏感性.

AIM： To establish gene-transfected cell strain L02/HBx and study its cell cycle changes.METHODS： Effectene transfection and G418 selection were used to obtain the positive clones of L02/HBx cells. Then HBx mRNA and protein expression were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot analysis, respectively. Finally, MTT assay and flow cytometry were adopted to measure the proliferation, apoptosis and cell cycles of L02/HBx cells.RESULTS： RT-PCR and Western blot analysis showed that the positive clones had HBx expression at mRNA and protein level. MTT assay demonstrated that the proliferation of L02/HBx cells had been accelerated. Flow cytometry found that the apoptosis rates of L02/HBx cells were at a lower level （0.09% ± 0.13% vs 3.74% ± 1.29%, P 〈 0.05）, and the proportion of L02/HBx cells fell G1 phase （61.35% ± 0.82% vs 67.80% ± 6.84%, P 〈 0.05） but rose in S phase （36.59% ± 2.54% vs 22.37% ± 2.17%, P 〈 0.05）. After coculture with adriamycin, L02/HBx cells manifested a higher apoptosis rate （34.91% ± 5.85% vs 0.09% ± 0.13%, P 〈 0.05）, and the proportion of Gl-phase cells was significantly increased （82.81% ± 6.48% vs 61.35% ± 0.82%, P 〈 0.05）, but still lower than that in the non-transfected group （82.81% ± 6.48% vs 87.19% ± 1.92%, P 〈 0.05）. However, the percentage of S-phase cells was markedly decreased （13.84% ± 6.16% vs 36.59% ± 2.54%, P 〈 0.05）, but still higher than that in the non-transfected group （13.84% ± 6.16% vs 2.22% ± 1.26%, P 〈 0.05）.CONCLUSION： L02/HBx cell strain stably expressing HBx is established successfully. HBx can accelerate the cell cycles and improve the growth instead of facilitating the apoptosis. L02/ HBx cells can be easily affected by the apoptotic factors, indicating that HBx may increase the susceptibility of normal liver cells to the apoptosis-inducing factors.