In vitro gene imaging by luciferase to detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand in lung cancer A549 cells

Abstract

Objective To detect the expression and effect of human tumor necrosis facctor related apoptosis-inducing ligand(hTRAIL)in vitro by using a novel double expressing adenoviral vector encoding hTRAIL and firefly lueiferase (luc) gene (Ad-hTRAIL-luc),in which luc wag used, as reporter gene.Methods A549 cells were transduced with the adenoviral vector encoding enhanced green fluorescent protein (EGFP) gene(Ad-EGFP)at variable multiplicity of infection(MOI).Adenoviral transducfion efficiency wag determined 48 h later.A549 cells were transduced with Ad-hTRAIL-luc at variable MOI.and the following tests were performed 48h later,respectively:the expressive ratio of hTRAIL and the apeptotic ratio of A549 cells were meagnred by flow eytometer;counts per minute(cpm)of luminescence were measurde by scintiUation counters. A549 cells were transduced with Ad-luc at variable MOI, and cpm of luminescence was measured by scintillation counters 48 h later. After A549 cells were transduced with AdhTRAIL-luc,the expressive ratio of hTRAIL,the apoptotic ratio of A549 cells and cpm of luminescence were analyzed by one-way ANOVA.The positive ratio of EGFP and cpm of luminescence (Ad-luc) were analyzed bv nonparametric ANOVA.Results After A549 cells were transfected with Ad-hTRAIL-luc,the expressive ratio of hTRAIL on the cell membrane of the groups were(2.37±0.04)/,(3.16±0.03)/,(3.64±0.03)/,(3.96±0.02)/,(4.24±0.02)/,(4.34±0.02)/ respectively,which showed significant difference between each other (P＜0.01);and the apoptotic ratio of A549 cells were (1.52±0.04)/,(2.93±0.02)/,(3.39±0.02)/,(3.64±0.02)/,(3.86±0.02)/,(4.08±0.02)/,(4.20±0.02)/,respectirely,and it showed significant difference between each other (P＜0.01);cpm of luminescence were 465 561±26 801,1 038 576±29 417,937 655±23 197,786 432±20 028,524 288±16 338,401 566±15 961,respectively,and it also showed significant difference between each other(P＜0.01).There was a positive relationship between the expressive ratio of hTRAIL and cell apoptotic ratio of A549 cells (r=0.984,P＜0.01).Conclusion The double expressing adenoviral vector Ad-hTRAIL-luc can transfer luc and hTRAIL gene to A549 cells efficiently,and the activity of luc may reflect the effect of hTRAIL as well as the expression of hTRAIL. Key words: Gene expression regulation; Receptors,yulnor necrosis factor; Gene therapy; Diagnostic imaging