BAX对Hep2细胞系的凋亡及化疗敏感性的影响

Effect of BAX on Apoptosis of Hep-2 Cells and Their Sensitivity to 5-Aza-CdR

目的:研究BAX基因对人喉鳞状细胞癌Hep2细胞系凋亡和对化疗药5-氮杂-2′-脱氧胞苷(5-Aza-CdR)敏感性的影响。方法:通过溴化二甲噻唑二苯四氨唑(MTT)比色法检测5-Aza-CdR对喉癌Hep2细胞体外增殖的影响。采用脂质体转染方法将BAX基因转入Hep2细胞后,应用RT-PCR法检测BAX基因在Hep2细胞中的表达。用流式细胞术检测Hep2细胞凋亡以及细胞周期变化情况。结果:通过用不同浓度的5-Aza-CdR处理喉Hep2细胞发现,5-Aza-CdR能抑制Hep2细胞体外生长,且呈时间与剂量依赖性,发现体外增殖的半数抑制浓度(IC50)为4μmol/L。通过流式细胞术检测发现5-Aza-CdR和BAX均可以使Hep2细胞发生G_1/S期细胞阻滞,并且能促进细胞凋亡。5-Aza-CdR处理后,与对照组相比,Hep2细胞G_0/G_1期的细胞数量明显增加,由对照组的51.57%增高到71.17%,凋亡率也由对照组的1.67%增加到13.96%。转染BAX后,RT-PCR结果显示转染BAX基因的喉鳞状细胞癌Hep2细胞中BAX表达显著增加(P<0.05),S期Hep2细胞百分比由空白对照组的33.29%、转染空载体组的32.42%分别减少至16.07%和13.58%,细胞凋亡率相对于空白对照组的1.67%、转染空载体组2.04%,转染BAX基因组的7.13%增加到23.74%,具有显著性差异(P<0.05)。本研究结果还表明同时转染BAX基因及加药组的喉鳞状细胞癌Hep2细胞凋亡率明显增加。结论:转染BAX基因能诱导Hep2细胞的凋亡,并能增加Hep2细胞对药物5-Aza-CdR杀伤作用的敏感性。

Objective： To investigate the effect of BAX on the apoptosis of human laryngeal squamous cell carcinoma Hep2 cells and their sensitivity to 5-aza-2＇-deoxycitydine （5-Aza-CdR）. Methods： The proliferation of Hep2 cells was detected by methyl thiazolyl tetrazolium （MTT） colorimetry after treatment with 5-Aza-CdR in vitro. The plasmid containing BAX cDNA was transfected into Hep2 cells. The mRNA level of BAX gene was detected by RT-PCR. Flow cytometry was used to analyze the apoptosis and cell cycle of Hep2 cells. Results： After treatment with different concentrations of 5-Aza-CdR, the proliferation of Hep2 cells was inhibited by 5-Aza-CdR in vitro in a dose- and time-dependent manner, and the IC50 was 4μmol/L. The results of flow cytometry detection showed that both BAX and 5-Aza-CdR could block Hep2 cells in G1/S phase and promote apoptosis. The percentage of cells in G0/G1 and the apoptotic rate of Hep2 cells treated with 5-Aza-CdR were increased from 51.57% to 71.17% and from 1.67% to 13.96%, respectively. The RT-PCR results showed that transfection of BAX significantly increased the expression of BAX gene level in Hep2 cells （P〈0.05）. After transfection, the percentage of Hep2 cells in S phase were decreased from 33.29% and 32.42% to 16.07% and 13.58% in blank control group and empty vector transfected group, respectively. The apoptotic rate of Hep2 cells transfected with plasmid containing BAX cDNA was increased from 7.13% to 23.74%, with a significant difference from that of the blank control group and empty vector transfected group （P〈0.05）. Conclusion： Transfection of BAX can induce apoptosis of Hep2 cells and increase their sensitivityto the killing effect of 5-Aza-CdR.