IFI16基因外来表达对喉癌细胞Hep-2的影响

Influences of IFI16 Gene Over-expression on Laryngeal Carcinoma Hep-2 Cells

将RT-PCR扩增得到的IFI16基因构建到真核表达质粒pVR1012中,得到pVR1012-IFI16重组质粒,用脂质体将其转染导入Hep-2细胞。半定量RT-PCR及Western blot检测IFI16基因在Hep-2细胞中的外来表达情况,细胞生长曲线绘制法及MTT法测定IFI16基因外来表达对Hep-2细胞增殖的影响,流式细胞术检测IFI16基因外来表达对Hep-2细胞周期及凋亡的影响。半定量RT-PCR分析结果发现,pVR1012-IFI16重组质粒转染的Hep-2细胞IFI16基因mRNA水平显著升高;Western blot分析结果发现,pVR1012-IFI16重组质粒转染的Hep-2细胞IFI16基因蛋白质表达水平显著升高;细胞生长曲线测定结果发现,第2天开始pVR1012-IFI16重组质粒转染的Hep-2细胞生长变慢,至第3天时pVR1012-IFI16重组质粒转染的Hep-2细胞生长速度明显变慢,与mock转染及空载体转染对照细胞相比差异显著(P<0.05);MTT测定结果发现,pVR1012-IFI16重组质粒转染Hep-2细胞48 h后,其相对活细胞数目显著降低,与mock转染及空载体转染对照细胞相比差异十分显著(P<0.01);流式细胞术检测结果发现,与mock转染及空载体转染对照细胞相比,pVR1012-IFI16重组质粒转染48 h后Hep-2细胞亚G0期细胞比例以及凋亡细胞比例都显著升高。上述研究结果说明,构建得到的pVR1012-IFI16重组质粒能在Hep-2细胞中外来表达IFI16基因,IFI16基因外来表达抑制Hep-2细胞增殖、阻滞Hep-2细胞周期在亚G0期并诱导Hep-2细胞发生凋亡。

IFI16 gene amplified from RT-PCR was constructed into eukaryotic expression plasmid pVR1012 to obtain pVR1012-IFI16 recombinant plasmid,and then this recombinant plasmid was transfected into Hep-2 cells with Lipofecter transfection.After transfection,the ectopic expression of IFI16 gene in Hep-2 cells was analyzed using semi-quantitative RT-PCR and Western blot methods,Hep-2 cells proliferation influenced by the ectopic expression of IFI16 gene was determined using cell growth curve drawing method and MTT method,and Hep-2 cell cycle and apoptosis influenced by the ectopic expression of IFI16 gene was analyzed using flow cytometry.Semi-quantitative RT-PCR result showed that the mRNA level of IFI16 gene was noticeably increased in pVR1012-IFI16 recombinant plasmid transfected Hep-2 cells,and Western blot result also showed that the protein level of IFI16 gene was obviously increased in pVR1012-IFI16 transfected Hep-2 cells.It was found from cell growth curve assay that Hep-2 cells grew slower than control cells after one day of transfection with pVR1012-IFI16 recombinant plasmid,and the growth rate of Hep-2 cells was obviously slower than control cells when transfected with pVR1012-IFI16 recombinant plasmid for two days and statistically different to that of control cells(P<0.05).MTT result showed that the relative viable cell number of Hep-2 cells was remarkably decreased after 48 h of transfection with pVR1012-IFI16 recombinant plasmid and significantly different to that of control cells(P<0.05 and P<0.01).Flow cytometry analysis disclosed that the ratios of sub-G0 phase cells and apoptotic cells in Hep-2 cells transfected with pVR1012-IFI16 recombinant plasmid for 48 h were obviously increased comparing to that of control cells.The above results illustrated that the pVR1012-IFI16 recombinant plasmid could ectopicly express IFI16 gene in Hep-2 cells and the ectopicly expressed IFI16 gene could inhibit Hep-2 cells proliferation,retard Hep-2 cells cycle in sub G0 and induce Hep-2 cells apoptosis.