线粒体DNA缺失HepG2细胞系的建立、鉴定及放射生物学特性

Observation of radiobiological characteristics in a HepG2 cell line with mitochondrial DNA deletion

目的为研究人肝癌细胞系Hep G2线粒体DNA(mt DNA)缺失后的放射生物学特性,建立并鉴定mt DNA缺失Hep G2(ρ0Hep G2)细胞系。测定辐射干预下mt DNA缺失(Rho0)肝癌细胞的凋亡情况、侵袭能力以及辐射敏感性的变化。方法在含有溴化乙锭(EB)、丙酮酸、尿嘧啶的特殊培养基中培养Hep G2细胞,经30次传代后,有限稀释法筛选完全去除mt DNA的克隆。去除丙酮酸及尿嘧啶后,观察细胞的存活情况。PCR法鉴定mt DNA的缺失。用6Mv X射线梯度剂量照射Hep G2细胞和ρ0Hep G2细胞,平板克隆法绘制生长曲线,线性二次方程拟合生存曲线,计算α/β。2Gy剂量辐射细胞,24 h后用Hochest33342细胞核染色,比较Hep G2细胞与ρ0Hep G2凋亡率的差异。用Transwell法测定两种不同细胞的侵袭能力。结果在含EB特殊培养环境下,Hep G2细胞可持续生长传代至30代,去除丙酮酸和尿嘧啶后,细胞短时间内大量死亡。经PCR法证实mt DNA完全缺失。ρ0Hep G2细胞α/β显著低于正常Hep G2细胞,辐射抵抗能力增强。辐射干预后ρ0Hep G2的凋亡比例显著减少。ρ0Hep G2穿膜细胞数显著增多。结论在EB长期诱导下,Hep G2肝癌细胞可被成功诱导为mt DNA缺失细胞。ρ0Hep G2细胞的辐射抵抗性显著增强,抗凋亡能力及侵袭能力均提高。

Objective To study the radiobiological characteristics of a Hep G2 cell line with mitochondrial DNA（mt DNA）deletion. Methods Hep G2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The Hep G2 cell line with mt DNA deletion（ρ0Hep G2 cells） were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mt DNA deletion in the cells was confirmed by PCR. The radiosensitivity of Hep G2 and ρ0Hep G2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X- ray exposure with Hochest33342 staining, and the invasiveness of ρ0Hep G2 cells was measured by Transwell assay. Results Hep G2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium.PCR confirmed total mt DNA deletion from ρ0Hep G2 cells, whose α/β value was significantly lower than that of Hep G2 cells.ρ0Hep- G2 cells showed an obviously lowered cell apoptosis rate following X- ray exposure with enhanced cell invasiveness.Conclusion Hep G2 cells can be induced by ethidium bromide into ρ0Hep G2 cells with an increased radiation resistance, antiapoptosis ability and cell invasiveness.