Gene Imaging By Luciferase In Vitro Detecing Therapeutic Effect Of HTRAIL For Lung Cancer A549 Cells

Objective: Applying double expressing adenovirus vector ad-luc-hTRAIL, in which luciferase was used as reporter gene, detected the expression of hTRAIL, and the expression of luciferase reflected the therapeutic effect of hTRAIL.Methods: 1 Transduction efficiency of adenovirus: A549 cells were transfected with ad-EGFP at different MOI. After incubation for 48h, by fluorescence microscope cells with positive expression of EGFP were observed and counted.2 Analysis of expression of hTRAIL: A549 cells were transfected with ad-luc-hTRAIL at different MOI. After incubation for 48h, adherent cells were collected and stained by indirect immunofluorescence labing method. The expression of hTRAIL were measured by flow cytometry.3 Assessment of growth inhibition: A549 cells were transfected with ad-luc-hTRAIL at different MOI. After incubation for 48h, cell viability was measured by using MTT assay.4 Detection of hTRAIL-induced apoptosis by FCM: A549 cells were transfected with ad-luc-hTRAIL at different MOI. After incubation for 48h, the survival cells were collected, labeled with Annexin V/PI assay kit, and then measured for the apoptotic rate with a flow cytometer.5 Detecting the expression of luciferase by scintillation counters5.1 A549 cells were transfected with ad-luc-hTRAIL: A549 cells were transfected with ad-luc-hTRAIL at different MOI. After incubation for 48h, lysis buffer was added to cover the adherent cells, and cells and all liqid were collected for high speed centrifugation. Then the supernatant was gained, and mixed with the luciferase assay reagent to detcte the expression of luciferase by scintillation counters .5.2 A549 cells were transfected with ad-luc: A549 cells were transfected with ad-luc at different MOI. After incubation for 48h, lysis buffer was added to cover the adherent cells, and cells and all liqid were collected for high speed centrifugation. Then the supernatant was gained, and mixed with the luciferase assay reagent to detcte the expression of luciferase by scintillation counters .Results: 1 Transduction efficiency of adenovirus: Transfected with ad-EGFP after 48h, fluorescence from cells was gradually up to strongest, and at the lowest MOI of 0.1, we can observe the positive cells. As the increasing of MOI, the positive cells with fluorescence were increasing. 99.5% of A549 cells were infected effectively by ad-EGFP at 100MOI. So the gene could be efficiently transducted by adenovirus to target cells.2 Transfected with ad-luc-hTRAIL at different MOI, cell viability was measured after 48h using MTT colorimetric analysis. Cell growth of each group was inhibited significantly (p<0.01) compared with the control group, and cell viability was gradually lower with the increase of MOI.3 By flow cytometry, we detected the expression of hTRAIL on the cell membrane of A549 cells, which had been transfected with ad-luc-hTRAIL, and the expression of hTRAIL was increasing as the increase of MOI; Meantime, we found the significant cell apoptosis compared with control (p<0.01) in A549 cells transfected with ad-luc-hTRAIL at different MOI, and the apoptosis rate was increasing as the increase of MOI; We found the expression of hTRAIL had positive correlation with the apoptosis rate of A549 cells (r~2=0.9939, p<0.01).4 By scintillation counters counts per minute were detected in each sample group of cells transducted with ad-luc-hTRAIL at different MOI. In the sample group of cells transducted with ad-luc-hTRAIL at MOI≤20,we found the counts per minute were increasing; but in the sample group of cells transducted with ad-luc-hTRAIL at MOI≥20, the counts per minute were decreasing, presumedly due to the enhancement of cell apoptosis rate induced by hTRAIL. It was at higher MOI that the activity of luciferase may better response to therapeutical effect of hTRAIL. And transfected with ad-luc after 48h, at the lowest MOI of 0.01, we could discover counts per minute by scintillation counters, and as the increasing of MOI, we also could detect increasingly counts per minute. So luciferase as reporter gene was very sensitive.Conclusion: 1 The double expressing adenovirus vector- ad-luc-hTRAIL could efficiently make the target gene transducted to A549 cells, and independently expressed hTRAIL and luciferase.2 The expression of hTRAIL induced by adnovirus produced significantly growth inhibition and contributed to cell apoptosis.3 With different dose of ad-luc-hTRAIL transducting A549 cells, the expression of luciferase can monitor the expression of hTRAIL, and efficiently reflect the therapeutical effect of TRAIL.