Study On The Relationship Between Oxidative Damage And Apoptosis Of Mdck Cells Infected By Influenza A Virus H1n1

BackgroundInfluenza is one common reason of seriousest impacts on human health. Since the beginning of the 21st century, H5N1 strain of bird flu and Mexican"swine flu"bring strong influence to the human work and life. In this way, the pathogenesis mechanism of influenza virus has been the focus of medical research. Oxidative damage mechanism and Influenza virus apoptosis are all important theories, but research about the relationship between the two theories haven't been reported. Other researches proved that oxidative stress could induce cell apoptosis through many pathways. Based on the research progress of influenza virus pathogenesis, this research formed a new perspective of the influenza virus into the relationship between apoptosis and cell oxidation during the period of infection.ObjectiveDuring the period of influenza virus infection and host cell apoptosis finally, the subject will study the role of oxidative damages in the pathogenic process. Namely, through the relationship research between oxidation damage degree and apoptosis,Evaluation the role of oxidative damage in the start-up, development and eventually pathogenic process of host cell apoptosis caused by influenza virus. Through this study, strengthen oxidative stress mechanism of the infected host cell apoptosis; provide a new idea for influenza virus pathogenesis mechanism research; provide new targets for the research of drugs against influenza; and provide a new perspective for the prevention and control of highly pathogenic avian influenza.MethodsPart one work out the experimental use dose of influenza virus. Inoculate influenza virus in Chicken embryos allantoic cavity and harvest virus. Test 50% tissue culture infective dose of MDCK cells and determine the experiment using dose of H1N1 influenza virus.Part two Test apoptosis rate of MDCK cell infected by influenza virus. MDCK cells were cultured and infected with H1N1 influenza virus. The apoptosis rates of the host cells were detected by AnnexinV-FITC/ PI double staining with flow cytometry after being infected for 0, 1, 3, 6, 12, 24 and 48 h.Part three Test MDA content of MDCK cell infected by influenza virus. MDCK cells were cultured and infected with H1N1 influenza virus. The MDA content of the host cells were detected by MDA testing kit after being infected for 0, 1, 3, 6, 12, 24 and 48 h. Part four Test the nucleic acid oxidation product of MDCK cell infected by influenza virus. MDCK cells were cultured and infected by H1N1 influenza virus. Through different treatments, the 8-oxo-G and 8-oxo-G expression content of the host cells'DNA and RNA were detected by cell climbing immunohistochemistry after being infected for 0, 1, 3, 6, 12, 24 and 48 h with special antibody.Results1. H1N1 influenza virus 50% tissue culture infective dose of MDCK cells was 10-4.5/0.1ml. Through experiment proved, the experiment using dose of H1N1 influenza virus was 100 TCID50/0.1ml.2. The experiment using dose of H1N1 influenza virus could obviously infect and induce cell apoptosis. The differences of the experimental groups'apoptosis rates were not more significant than the control groups'for the first 6 hours (P>0.05), but the 12, 24 and 48h groups'had significant high apoptosis rates than the corresponding control ones'and the former 4 experimental groups, rising to 6.422±0.326%, 9.570±0.229% and 9.178±0.286% respectively (P<0.01).3. MDA test showed that its amount was declining with the time going. The MDA amount reach the highest level (1.490±0.053nmol/mgprot) when the cells were infected(P<0.01), and maintain high balance (1.221±0.036 nmol/mgprot,1.157±0.061 nmol/mgprot,1.089±0.038 nmol/mgprot) at 1, 3, 6h(P<0.01). the amount obviously decline to 0.636±0.033 nmol/mgprot at 12 h(P<0.01), and then, no statistical significance differences could be observed between the experimental groups and the corresponding control ones at 24, 48h(P>0.05).4. MDCK cells were cultured and infected with H1N1 influenza virus, DNA 8-oxo-G expression positive cells percentage increased significantly with the time going (P<0.05). The positive cells percentage reached 12.20±3.16 at 0h (P<0.05). with significant differences between the experimental groups'and the control groups'at 1h,3h,6h,12h,24h(P<0.01),reaching the highest point 87.45±3.98 at 48h(P<0.01). RNA 8-oxo-G positive cells percentage increase significantly with the time going (P<0.05). the positive cells express percentages respectively are 9.93±1.45;12.38±1.92;26.69±0.86;46.55±2.92;58.89±2.02;72.72±2.13;80.63±0.97 for 0, 1, 3, 6, 12, 24, 48h(P<0.01). ConclusionIn this study, we found out that:The host cells appeared typical cell pathological effects and death after infected by H1N1 influenza virus using the experimental dose. With the time going, the apoptosis rates increased apparently, MDA results exposed that the host cell membrane lipid damages had happened in the early stage after infection, and the lipid oxidative damage degree declined with time going. 8-oxo-G is the products of DNA guanine nucleoside caused by oxidative stress; it was also the most important mark of DNA oxidative damage. Through examining 8-oxo-G expression rates of RNA and 8-oxo-G expression rates of DNA, we got that oxidative stress damage not also the cell membrane lipid but also the nucleic acids. So we concluded, oxidative damages existed all the stages of the infection and apoptosis of the host cells. Oxygen free radicals invaded and induced membrane lipid oxidative damage products firstly, and the host cell nucleic acids were damaged continuously,the damage influenced cell transcription, translation hardly.