| Hantaan virus is the pathogeny of hemorrhagic fever with renal syndrome (HFRS).It multiplicates in primary and passaged cells, but there is few cellular pathogenic effect in these infected cells.Researches show that integrin may be the cellular membrane receptor for hantaan virus and it is related to hantaan virus's entry. In addition, hantaan virus causes changes of morphology and metabolism et al in infected cells. Furthermore, it can induce apoptosis in human embryonic kidney cell and lymphocyte, but the mechanism is not clear. Study the effect of hantavirus on primary human embryonic pulmonary fibroblasts (HEPFs) will get evidence for the molecular mechanism of interaction between hantavirus and host cells. The development of gene chip brings forth new area for investigating the interaction between pathogens and host cells, and expression microarray can answer how host cells or organ to recognize pathogens, in which level the host can distinguish different pathogenic factors and indicate the cross talk between host and pathogens, potential therapeutic target and the prognosis mark for disease. In thus, dynamic analysis of the level of gene and related protein expression and cell cycle in HEPFs infected with hantavirus through techniques of gene chip and flow cytometry and by using inhibitor of mRNA-cordycepin, will add a new page into the study of interaction between hantavirus and cells.Objective: To analyze the gene expression profile, activity of caspase 3, phosphatidylserine (PS) translocation and change of cell cycle of HEPFs infected with Hantaan virus (HTNV) 76-118 dynamically,and to learn the effect of hantavirus on HEPFs and apoptosis induced by hantavirus.Methods: 1. Experiment groups and virus inoculation HEPFs were classified as three groups randomly. They were virus infection group, virus infection with cordycepin treatment group and normal control group. Cells in former two groups were infected with hantaan virus (M.O.I. 5-10),after inocubated in 37C for 2 hours, medium of infection and control group were changed with 5% maintaining DMEM, and medium of infection withcordycepin treatment group were changed with 5% maintaining DMEM with 200 u M cordycepin.2. Observing HEPFs infected with hantavirus HEPFs were investigated directly by phase-microscope after hantavirus infection with or without cordycepin treatment at 6 hour (h), 24h and 96h post infection (p.i.) respectively. Furthermore, cells smears were prepared for the detection of viral antigen of different cells in different group by indirect fluorescent assay (IFA).3. Total RNA extraction from HEPFs in different group HEPFs were harvested at 6 h, 24h and 96h p.i. with or without cordycepin treatment. Then total RNA were extracted by modified one-step method and the quality of RNA was also analyzed.4. Detection of viral nuclear acid Total RNA of each group was analyzed by reverse transcription-polymerase chain reaction(RT-PCR) for detecting the nuclear acid of HTNV.5. Choosing the differently expressed genes by gene chip The intact cellular RNAs of each group were used for reverse transcription as well as labeling by fluorescence. Then experimental groups were labeled by Cy5 and control groups were labeled by Cy3. All these labeled cDNAs were hybridized with chips which were used in our study. After hybridization, chips were scanned and fluorescent signals were analyzed for choosing variational genes.6. Detection of the activity of caspase 3 Duplicate cell plates were set up for the following samples: hantavirus infection group, hantavirus infection with cordycepin treatment group, negative control group, hantavirus infection with inhibitor for caspase 3 and hantavirus infection without substrate for caspase 3. Cells of each group were harvested and were resuspended in chilled cell lysis buffer. Then the supernatants of each group were harvested and following processes were done according to BD ApoAlert Caspase 3 assay's user handbook. The data were read at 405 nm in a microplate reader and units of caspase... |
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