摘要
目的研究乙型肝炎病毒X基因(HBX基因)对HL7702肝细胞凋亡及凋亡相关因子表达的影响。方法用脂质体转染法将HBx真核表达载体pcDNA3.1(+)-X瞬时转入HL7702细胞,以转染空质粒pcDNA3.1(+)的细胞及未转染的HL7702细胞为对照。转染后72 h收集细胞标本,作以下处理:RT-PCR法检测HBXmRNA的表达,免疫荧光鉴定HBx蛋白的表达;流式细胞术检测细胞凋亡率;以β-actin为内参,半定量RT-PCR法检测凋亡相关基因Bax、Bcl-2 mRNA的表达量变化。结果转染72 h后,转染pcDNA3.1(+)-X真核表达载体的HL7702细胞经RT-PCR扩增出HBX片段,免疫荧光结果显示细胞内出现HBx蛋白的较强荧光;而转染空质粒组及未转染对照组经RT-PCR法及免疫荧光检测均显示无HBx表达。通过流式细胞术和半定量RT-PCR法检测细胞凋亡情况,结果显示:转染HBX的细胞凋亡率(3.38%±0.67%)相对于转染空质粒组(1.25%±0.30%)及未转染质粒组(1.40%±0.37%)凋亡率增高,差异均有显著性意义(P<0.05);转染HBx的细胞Bax mRNA相对表达量较转染空质粒组和未转染质粒组明显增高,差异均有显著性意义(P<0.05),Bcl-2 mRNA相对表达量较转染空质粒组和未转染质粒组明显降低,差异均有显著性意义(P<0.05)。转染空质粒组和未转染质粒组之间,对细胞凋亡率、Bax mRNA、Bcl-2 mRNA进行比较,差异无统计学意义(P>0.05)。结论成功将HBX转染入HL7702细胞,并在细胞中表达;转染后72 h HBx可上调Bax表达,而下调Bcl-2表达,表明HBx能促进HL7702细胞凋亡。
Objective To investigate HBx＇s effect on the apoptosis of HL7702 and the expression of apoptotic factors. Methods The HBx gene eukaryon expression vector pcDNA3.1 （ ＋ ） - X was transiently transfected into HL7702 cells by lipid - media transfection ,with HL7702 transfected with pcDNA3.1 （ ＋ ）and HL7702 untransfected taken as controls. 72 h after transfection ,the cells were collected to proceed with the following tests ： expression of HBX mRNA by RT -PCR,expression of HBx protein in cells by immunofluorescency, apoptotic rate by flow cytometry, evaluation of the mRNA levels of Bax and Bcl - 2 by semi - quantified RT - PCR using β - actin as an internal control. Results HBX mRNA was detected in HL7702/pcDNA3.1 （ ＋ ） -X cells by RT -PC ,showing strong immunofluorescence;whereas no expression of HBx was detected in the controls. As compared with the untransfected HL7702 group （ 1. 40% ± 0.37% ） and HL7702/pcDNA3.1 （ ＋ ） group （ 1.25% ±0.30% ） ,the apoptotic rate in HL7702/pcDNA3.1 （ ＋ ） - X group was obviously increased （3.38%± 0.67% , P 〈 0.05 ）. Semiquantitative RT- PCR analysis showed Bax mRNA level was significantly higher in HBX transfection group than those in the controls （ P 〈 0.05 ） , whereas the Bcl - 2 mRNA level was significantly lower in he HBX transfected group than in the controls （ P 〈 0.05 ）. No significant differences in apoptotic rate, Bax mRNA level or Bcl -2 mRNA level were found between the two control groups. Conclusion HBX gene can be transfected into HL7702 cells successfully. HBx up - regulates Bax and down - regulates Bcl - 2, suggesting HBx can induce liver cell apoptosis.
出处
《徐州医学院学报》
CAS
2008年第4期217-221,共5页
Acta Academiae Medicinae Xuzhou
基金
江苏省教育厅课题(07KJD310220)
徐州市课题(XM07C056)
徐州医学院课题(07KJ41)
