摘要
目的探讨重组质粒pEGFP-N2/XPD的转染条件和Pifithrin-α（P53抑制剂）的孵育条件。方法利用脂质体将质粒pEGFP-N2/XPD在不同质粒浓度时转染细胞以及转染后分别孵育不同时间,然后进行RT-PCR和Western blot检测着色性干皮病基因D（XPD）的表达。将Pifithrin-α在不同浓度时孵育细胞以及分别孵育细胞不同时间,然后进行MTT检测。结果 XPD的表达在质粒浓度为0~4μg·孔-1范围内呈剂量依赖性升高（P〈0.01）,而质粒浓度达到4μg·孔-1以后XPD的表达不再随浓度的增加而继续升高;转染后细胞孵育时间为48h时XPD mRNA的表达为最高（P〈0.05）,转染后细胞孵育时间为72h时XPD蛋白的表达为最高（P〈0.01）。细胞增殖活力在Pifithrin-α浓度为0~20μmol·L-1范围内呈剂量依赖性升高（P〈0.05）,而Pifithrin-α浓度达到20μmol·L-1以后细胞增殖活力不再随浓度的增加而继续升高;细胞增殖活力在Pifithrin-α孵育时间为0~24h范围内呈时间依赖性升高（P〈0.01）,而Pifithrin-α孵育时间达到24h以后细胞增殖活力不再随孵育时间的增加而继续升高。结论在后续的研究中,将以浓度为4μg·孔-1的pEGFP-N2/XPD质粒转染HepG2.2.15细胞,转染后的第2天以浓度为20μmol·L-1的Pifithrin-α孵育细胞24h,转染后共孵育细胞48h。
Objective To explore the transfection of recombinant plasmid pEGFP-N2/XPD and the incubation of pifithrin-α（inhibitor of P53）.Methods Cells were transfected with different concentrations of plasmid pEGFP-N2/XPD by liposome and were incubated for different time periods.The expression of XPD was detected by RT-PCR and Western blot.In addition,cells were incubated with different concentrations of pifithrin-αfor different time periods.Cell viability was measured by MTT.Results The expression of XPD was significantly increased by transfection with pEGFP-N2/XPD（0-4μg per well）in a dose-dependent manner（P＆lt;0.01）.The maximal effect was observed at the concentration of 4μg per well.Moreover,the highest expression of XPD mRNA and protein was found after incubation for 48and 72hours,respectively（P＆lt;0.05 and P＆lt;0.01,respectively）.Cell vitality was significantly increased by incubation with pifithrin-α（0-20μmol·L-1）in a dose-dependent manner（P＆lt;0.05）.The maximal effect was observed at the concentration of 20μmol·L-1.Furthermore,cell vitality obviously increased with time（P＆lt; 0.01）.However,cell viability did not further increased after pifithrin-αincubation for more than 24hours.Conclusion In subsequent experiments,HepG2.2.15cells will be transfected with pEGFP-N2/XPD（4μg per well）and incubated with pifithrin-α（20μmol·L-1）at day 2after transfection for 24hours.Altogether 48hincubation cells after transfection.
出处
《南昌大学学报（医学版）》
CAS
2013年第8期1-5,共5页
Journal of Nanchang University：Medical Sciences
基金
国家自然科学基金(81300348)
江西省教育厅科学技术研究项目(GJJ13158)
