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

Author: LiuHaiBin
Tutor: FanJingPing；LiaoJianChun；WuJian；LinShunZhang；WangHaiQing
School: Second Military Medical University
Course: Department of Otolaryngology Head and Neck Surgery
Keywords: Recombinant adenovirus mda-7/IL-24 Laryngeal Apoptosis Detection
CLC: R739.65
Type: Master's thesis
Year: 2010
Downloads: 30
Quote: 0
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Abstract

[Objective] 1. Construct recombinant adenovirus carrying the gene] mda-7 (melanoma differentiation-associated gene-7, melanoma differentiation associated gene -7) / IL-24 (Interleukin-24, interleukin-24). 2. Constructed vector system Ad-mda-7/IL-24 people of Hep-2 cell lines after infection by qualitative and quantitative methods to observe the impact of laryngeal cancer cell growth. [Method] 1. The identification pPEP4 plasmid mda-7/IL-24 KpnI and XhoI restriction endonuclease enzyme digestive gland virus shuttle the plasmid vector pAdtrackCMV (containing green fluorescent protein GFP) to transform competent E.coli connected DH5a, kanamycin clone was extracted plasmid, digestion, PCR identification; the PmeI single was linearized carry mda-7/IL-24 gene the shuttle plasmid pAdTrackCMV-mda7, adenovirus backbone plasmid AdEasy-1 together electrically transformed into competent cells recombinant dedicated BJ5183 two plasmids homologous recombination, and kanamycin streptomycin double antibody clones were screened, plasmid extraction, electrophoresis, PacI digested recombinant cloning, the positive clones the plasmid was then transformed into E.coli DH5a competent, kanamycin clones were screened again extracted plasmids, electrophoresis, restriction analysis, PCR identification the pAd-mda7 and pAd-GFP; restriction endonuclease the enzyme PacI the single cut the pAd-mda7 and pAd- GFP, cut plastic recycling plasmids quantitative transfected human embryonic kidney 293T cell line virus amplification, the collected viral titer was determined in parallel alternate. 2.Hoechst33258 dyeing --- qualitative detection: of Hep-2 cells were seeded in 6-well plates, a hole counts, three holes were added to the same amount must multiplicity of infection (MOI) of Ad-mda-7 (Ad group) Ad-GFP (Ead group) and a PBS buffer solution (Control group). 48h after infection, the cells were fixed by Hoechst33258, after repeated washing inverted number of cells was observed under a fluorescence microscope, and cell morphology and cell color distribution and brightness. 3.PI staining, flow cytometry analysis - quantitative detection: Select the appropriate multiplicity of infection (MOI) infected with Hep-2 cells in 6-well plates quantitative vaccination 48h after the cells were collected by centrifugation, PBS buffer diluted, added ten thousandths III TritonX-100 cell membranes punch, the working concentration of PI (propidium iodide) dark staining, flow cytometry detection, determination of cell cycle. [Results] 1. Use of replication-defective adenovirus vectors to successfully build Ad-mda-7/IL-24 well Ad-GFP. 2 observed under the microscope: 24h after the Ad-mda7 group and Ad-GFP group Hep-2 cells beginning of detachment, and the Ad-mda7 group compared with Ad-GFP group out of more and less the total number of cells. Normal growth of the PBS-treated group, only a small number of cells detachment; 48h after the Ad-mda7 group significantly affect the growth state, poor state, adherent cells decreased significantly affected to some extent, and the Ad-GFP group cell growth state, part of the growth of cells from the wells , compared to the former, the number of cells, more (still adherent cells and cells of the total number), the growth of the state better. PBS cells has been basically covered with the the wells naturally out of the growth of a small number of cells. 72h after cell growth status obviously deteriorated Ad-GFP group the Ad-mda7 group out of a large number of the PBS treated many cell detachment. 3. Observed by fluorescence microscopy of Hoechst 33258 apoptosis staining results: the Ad-mda7 treated cells pyknotic cell nuclei dense stain color white; Ad-GFP treated cell contraction and about 20% of the cell detachment, but no the apoptosis staining characteristics; PBS-treated cells form full and normal growth. 4.PI staining flow cytometry results: MOI = 30 infections after 48h the Control group SubG1 phase was 5.84%; the Ead group SubG1 period 14.53% 21.65%; the Ad-mda7 processing, SubG1 phase was to apoptotic cells proportion of incremental; MOI = 50, the G2M Block test results: the Ad-mda7 group G2 / M Block period the proportion of 40.60% in the Ad-GFP group G2 / M Block period was 7.93%, the PBS group corresponding G2 / M Block period ratio of 2.24%. [Conclusion] 1. Recombinant human adenovirus system (by shuttle plasmid pAdTrackCMV adenovirus backbone plasmid AdEasy 1, as well as restructuring optimization coli BJ5183 composition), build green fluorescent protein (GFP) and polyclonal area shuttle plasmid pAdTrackCMV , can be used mounted mda-7/IL-24. The backbone plasmid AdEasy 1 deletion of the E1 region and the E3 region of the adenoviral genome, making the production of adenovirus can not be self-amplified in the cells other than 911 and 293, to ensure the controllability of the dose of the experimental and biological safety, but retaining the virus efficient ability to infect and equipped with the ability of the gene protein expression. 2.Ad-mda-7/IL-24 transfection laryngeal hep-2 cells after 48h better detection indicators 72h cell growth status obviously deteriorated cells out of each experimental group, and is not suitable for further cell processing and detection. 3 adenovirus carrying mda-7/IL-24 on laryngeal hep-2 cell growth inhibition and pro-apoptotic role blockade and laryngeal cancer cell growth may be positively correlated with the multiplicity of infection.

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