Study On The Oxidative Damage Mechanism Of MDCK Cells Infected By Influenza A Virus H1N1

BackgroundInfluenza infection is one common factor that seriously impacts human health. 1930 R.E.Shope successfully isolated flu virus by infecting ferrets with the swine respiratory filtered liquid[1]. 1933 Smith first named the virus isolated from the human body influenza A virus [2]. The first influenza pandemics caused 2000-4000 million deaths in 1918 -1919 [3,4]。In the beginning of this century, the 2009 H1N1 influenza viruses attacked human and caused a world-wide pandemic. The pathogenesis of influenza virus infection has been the focus of medical research.In 2010,LiuYan-ming [5] use Immunohistochemistry method to investigate expression of biochemical marker 8-hydroxy-2′-desoxyguanosine (8-OXO-dG) of DNA damage in MDCK cell after infected with H1N1 influenza virus.Results indicated that H1N1 influenza virus infection can induce significant DNA oxidative damage.Oxidative stress not only caused oxidation of protein and lipid, but also made nucleic acids be oxidated. In another study, Results showed that H1N1 influenza virus can significantly induce apoptosis of the host MDCK and with the time going, the apoptosis rates increased apparently [41]. The guanine can be oxidized in situ, or replaced into genome in the form of 8-oxo-dG during DNA replication. In human cells, the maintenance of a low oxidation state depend on multiple gene repair enzymes. For example, MTH1 can hydrolyze 8-oxo-dGTP to 8-oxo-dGMP, and block error incorporation process [7]; OGG1 can remove 8-oxo-dG that is formed in situ or incorporated during DNA replication from genome [8]. The two enzymes cooperate in different stages to prevent or reduce oxidative damage of DNA, so that the normal genome DNA can be maintained in a low oxidation state. Published in DNA Repair in February 2008, Hill found that over-oxidation of DNA in cultured cells was related to the degradation of OGG1[9]. Our preliminary results indicate that the quantity of 8-oxo-dG within target cells would increase after infected with influenza viru, which suggested that oxidative stress ultimately lead to apoptosis of target cells. According to the research progress, we believe that Oxidative damage repair enzymes participate over-oxidation DNA repair processe, this may play a role during the apoptosis of host cells infected by influenza virus. It has been rarely reported that the mechanisms of DNA oxidative damage of host cell infected with influenza in the current international. This research will explore influenza virus pathogenesis from view ofDNA oxidative damage repair.ObjectiveThe subject aims to further explore the possible pathogenic mechanism of H1NI virus, on the base that influenza virus infection can cause lipid oxidation and DNA oxidative damage of the host cells. Evaluate the role of oxidative damage in the process of host cell apoptosis caused by influenza virus. The expression of DNA repair enzyme is not enough to fight the excessive oxidative damage, eventually which leadto host cell apoptosis and the occurrence, development of symptoms. The aim of this study is to clarify oxidative stress mechanism of the infected host cell; provide a new idea for influenza virus pathogenesis mechanism research; new targets for the research of drugs against influenza; and a new perspective for the prevention and control of highly pathogenic avian influenza.MethodsPart one Set up Influenza virus infection cell modelMDCK cell were cultured using cell culture technology. 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 Study on the expression of MTH1 gene in MDCK cells infected by influenza A virusEstablishment of a semi-quantitative RT-PCR system for detection of MTH1 expression:Total RNA within cultured MDCK cells were extracted and used for RT-PCR. The expression quantity of MTH1 and control gene GAPDH were analyzed using Scion Image software and then the ratio of MTH1/ GAPDH were used for the accuracy, repeatability and sensitivity analysis of the method. MDCK cells were cultured and infected with H1N1.The relative expression of MTH1 gene of the host cells were detected by RT-PCR after infected for 1,3,6,12,24 and 48 hours.Part three the expression of OGG1 gene in MDCK cells infected by influenza A virusRNA was extracted from cultured MDCK cells, then reverse-transcripted into cDNA, and design primers for polymerase chain reaction amplification of the target gene OGG1. The expression quantity of MTH1 and control gene GAPDH were analyzed with Image J software and then the ratio of OGG1/ GAPDH were used to analyse teh the accuracy and repeatability of this method. MDCK cells were cultured and infected with H1N1.Then, the relative expression of OGG1 gene of the host cells were detected by RT-PCR after infected for 1,3,6,12,24 and 48 hours.Results1.MDCK cells and H1N1 influenza virus were successfully cultured. H1N1 influenza virus 50% tissue culture infective dose was 10-3.91/0.1ml. The experiment using dose of H1N1 influenza virus was 100 TCID50/0.1ml.2.(1) Sequencing results of the RT-PCR products show identical with that of the GenBank. (2) Sensitivity detection revealed this system could detect the positive expression of MTH1 from only 50ng total RNA. (3) Repeatability detection showed that the averaged ratio of MTH1/ GAPDH was 2.02±0.09 (n=10), and the CV was 4.31%. There were no significant differences of MTH1 expression between the experimental groups and the control groups at 6 and 12 hours after H1N1 infection,but the MTH1 expression was significantly higher than that of the control group at 1 h and 3 h, and was significantly lower than that of the control group at 24 h and 48 h after H1N1 infection. 3.Primer can amplify the reference gene and target gene. The sequencing of the RT-PCR PCR products was consistent with the OGG1 gene sequence derived from the Gene Bank. Repeatability detection showed that the averaged ratio of OGG1/ GAPDH was 0.84±0.025 (n=6), and the CV was 2.99%. There were no significant differences of OGG1 expression between the experimental groups and the control groups at 48 hours,but the OGG1 expression was significantly higher than that of the control group after at 1h,3h,6h,12h and 24h of infection.ConclusionIn this study, we found the length of amplified fragment was consistent with the anticipation, and the sequence of that matched with the GenBand sequence. There was no positive amplification in negative control, which confirmed the specificity of this method. Repetitive testing results identified the good repeatability of the system. In summary, the system has not only excellent accuracy, high sensitivity and repeatability, but also low equipment requirement and easy operation. The establishment of this system can lay a good foundation for the research of MTH1 mRNA expression and influenza virus infection related research. RT-PCR detection of MTH1 gene expression indicated that mRNA levels of MTH1 gene continued to increase from 1h to 3h, and the levels reduced to normal from 6h to 12h, after H1N1 influenza virus infected such cells. At the monitoring points of 24h and 48h, MTH1 expression was significantly lower than that of the control group. MTH1 could prevent DNA from oxidative damage, which indicate that target cells timely increase MTH1 expression to strengthen the antioxidant capacity of themselves in early stages that oxidative damage appear. While in the later stages of infection, cells apoptosis increased and then the expression of MTH1 was inhibited, so DNA oxidative repair capacity was decreased. With deepening of viral infections and increasing of oxidative stress products, the death of infected cells also increased correspondingly. Method study results showed the length of amplified fragment is consistent with anticipation, and sequencing of such fragment is also identical with GenBand sequence. After H1N1 influenza virus infected cells, mRNA levels of OGG1 with target cells continued to increase from 1h to 24h, whicn can compensate cells to repair DNA oxidative damage, then reduced from 48 hours, and at this time cells apoptosis factors may happen heavily because of the decreasement of OGG1 expression. The change of expression of OGG1 may play a role in influenza pathogenic mechanism. In 2009, Ke found that the mRNA level of hOGG1 in pulmonary type_II_like epithelial cells A549 also increased dramatically under H2O2 exposure. This study explores the influenza virus from aspect of oxidative stress and deepens the understanding of oxidative stress mechanism of influenza virus-induced host cells.