Adenovirus-mediated Human Mda-7/IL-24 Gene Transfer Induces Apoptosis In Human Laryngeal Carcinoma Cell Line Hep-2

[Objective]1.Construction of adenoviral vector carrying the gene mda-7 (melanoma differentiation-associated gene-7)/IL-24 (Interleukin-24).2. After infected human laryngeal carcinomaHep-2 cells with ad-mda-7/IL-24, adopting qualitative and quantitative approaches to observe of the laryngeal carcinoma cell growth.[Methods]1. Identification pPEP4 plasmid with mda-7/IL-24, KpnI and XhoI restriction endonuclease cleaved adenovirus shuttle vector pAdtrackCMV (containing green fluorescent protein GFP), then transformed into E.coli DH5a, kanamycin selection cloning, plasmid extraction, restriction enzyme digestion, PCR identification; PmeI single-digested linearized the shuttle plasmid carrying mda-7/IL-24 (pAdTrackCMV-mda7), and the adenovirus backbone plasmid AdEasy-1 and the shuttle plasmid were transformed to BJ5183 for homologous recombination, through kanamycin and streptomycin double-resistant screening cloning, plasmid extraction, electrophoresis; PacI digested recombinant single clone, the positive clones were then transformed into E.coli plasmids DH5a Competent, kanamycin clones, plasmids extracted again, electrophoresis, restriction enzyme digestion, PCR identification and got pAd-mda7 and pAd-GFP; restriction endonuclease PacI single-cleaved pAd-mda7 and pAd-GFP, cleaved plastic recycling. Quantitative plasmids were transfected to human embryonic kidney cell line 293T virus for amplifying and collected the virus for tittering.2. Hoechst33258 staining---qualitative detection:laryngeal carcinoma Hep-2 cells were seeded in 6 well plate, a well for counting, three wells of which were added to a certain multiplicity of infection (MOI) of Ad-mda-7 (Ad group), Ad-GFP (Ead group) and PBS buffer solution (Control group) equally.48h later cells were fixed and stained with Hoechst33258 after repeatedly washed and Observe the cell number, cell morphology, cell distribution and brightness of color by inverted fluorescent microscope.3. After PI staining counted by Flow Cytometry---quantitative detection:Select the appropriate multiplicity of infection (MOI) and infected quantitative inoculation of Hep-2 cells in 6-well plate. After 48h the cells were collected, centrifugated, PBS buffer diluted and then added three ten thousandths of Triton X-100(the working concentration) to the wells for membrane punching. Flow Cytometry counts were detected in cell cycle.[Results]1. Ad-mda-7/IL-24 and Ad-GFP was successfully constructed by using replication-defective adenoviral vector.2. Observed under light microscope:after 24h, Hep-2 cells in Ad-mda7 and Ad-GFP group began to detach from the well, and they are more in Ad-mda7 group than in Ad-GFP group of the total number of cells. PBS treated group (control group) showed normal growth, only a few cells detaching away; after 48h, Ad-mda7 group significantly affected state of the growth, poor state, and attached cells decreased; whereas the cell growth state of Ad-GFP group was affected to some extent, part of the cultured cells from the well growth compared to the former, the growth state of most cells is in better shape. Cultured cells in PBS group have been basically covered the well, though a small amount of cells detached naturally. After 72h the cell state was obviously worse, there are a large number of cells detaching in Ad-GFP group and Ad-mda7 group. At same time, in PBS treatment group, are there many cells detaching away.3. Apoptosis observation using Hoechst 33258 staining by fluorescence microscope: Ad-mda7 treated cells was condensed, dense nuclear stain, the color white; Ad-GFP treated cells was also some shrinkage and 20% cell detachment, but no apoptosis staining characteristics; PBS treated cells showed normal growth.4. PI staining Flow Cytometry results:(MOI= 30) The proportion of Control Group SubGl after 48h is the proportion of 5.84%; Ead group SubGl 14.53%; and Ad-mda7 treatment group, the ratio of SubGl is 21.65% and it is an increasing proportion; G2M Block Test Results:The proportion of Control Group SubGl after 48h is the proportion of 2.24%; Ead group 7.93%; and Ad-mda7 treatment group, the ratio of G2/M Block is 40.60%.[Conclusion]1. Recombinant human adenovirus 5 system includes a shuttle plasmid pAdTrackCMV, an adenovirus backbone plasmid AdEasy 1, and re-optimize the composition of E. coli BJ5183. The shuttle plasmid pAdTrackCMV built with green fluorescent protein (GFP) and the polyclonal region can carry mda-7/IL-24. The backbone plasmid AdEasy 1 with deletions of E1 and E3 zone area, making the production of adenovirus can not be self-amplified other than in 911 or 293 cells to ensure experimental control and the dose and biological security, but retains the ability to virus infection and efficient expression of genes carrying capacity.2. after 48h Ad-mda-7/IL-24 transfected laryngeal carcinoma cell line hep-2, detection of the target is better; Cell growth state for 72h was obviously worse, so a large number of cells from each experimental group are not suitable for further cell processing and testing.3. Adenovirus carrying the mda-7/IL-24 induces laryngeal carcinoma cell hep-2 growth inhibition and apoptosis, and the block on the role of laryngeal carcinoma cells may be correlated with the multiplicity of infection.