| Respiratory syncytial virus(RSV) is the principal viral pathogen associated with acute lower respiratory infections in infants and children. It seems that there is a strong link between RSV bronchiolitis and asthma in epidemiology and immunology. The pathogenesis of RSV infection has not been fully understood .The effective therapy and vaccines are not available at present. DNAzyme (deoxyribozyme) is another novel molecular biological tools following the ribozyme,which could cut the RNA substrate between a unpaired purine and a paired pyrimidine residue.Thereby DNAzyme could suppress the expression of message RNA.The purpose of this study is to investigate the expression of ICAM-1 and the activity of nucleic factor-κB in RSV infected epithelial cells.We also designed and synthesized a special 10-23 deoxyribozyme against the M2-2mRNA of respiratory syncytial virus to investigate its potent ability of the inhibition of RSV replication in cultured cells.Part 1:The changes of intercellular adhesion molecular and nucleic factor-κB in respiratory syncytial virus infected epithelial cellsResearch background and Objiective Intercellular adhesion molecule -1 (ICAM-1) is a important biological active agents which plays a key role on the emergence and development of local inflammation. It is the only known ligand on epithelial cells for neutrophils binding. ICAM-1 is expressed on a variety of cells including endothelial cells,fibroblasts and epithelial cells.The respiratory epithelium is not only a physical barrier between environmental noxious agents and the internal milieu but also a metabolically active physiochemical structure. Respiratory epithelial cells are both target and effector cells in airway inflammation. Epithelial cells are able to express adhesionmolecules ,such as intercellular adhesion molecules-1,and to produce a wide array of cytokines.Thereby the epithelial cells are involved in inflammation and immune response. Respiratory syncytial virus is the most frequent cause of acute lower respiratory infection and pneumonia in infants requiring hospitalization. Although it has been reported that RSV infection upregulates ICAM-1 expression on epithelial cells, it is unclear whether the RSV infection could induce the upregulation of ICAM-I on 9HTE cells in a time-dependent pattern. The study reported here were designed to establish the cell model of RSV infection in vitro, and then to test the expression of ICAM-1, the binding activity of NF-κB in 9HTE cell lines at different time after RSV infections. Nuclear facror-κB are able to increase the expression of inflammation factor which genens contain the binding sites of NF-κB.To test whether the RSV infection can increase the expression of ICAM-1 on 9HTE cell and the role of NF-κB in epithelial cell inflammation induced by RSV, the first we eststablish the cell model of RSV infection in vitro,then we analysis the changes of ICAM-1 expression and the activation of NF-κB B in 9HTE cell infected with RSV .Methods:The cytopathogenic effect induced by RSV with various multiplicity of infection(moi) was observed directly.The expression of ICAM-1 protein on 9THE cells infected with 0.1 moi RSV were detected by inmmunohistochemistry stain.We also take the methods of RT-PCR to detected the changes of ICAM-1 mRNA on 9HTE cell at 0.5h,1h,2h and 3h respectively after RSV infections.The analysis of nuclear factor -κB was measured by electrophoretic mobility shift assay(EMSA).Results:The cell model of RSV infection was established in vitro successfully . The epithelial cells infected with RSV mostly survived after 5days cell culture when the multiplicity of infection was above 0.001 , nevertheless, the 9HTE cells almost died when the multiplicity of infection is above 0.1. The results of immunohistochemistry stain showed that the intercellular adhesion molecule -1 protein was expressed on non-infected 9HTE cells in a low level and obviously increased at 48hrs after RSV infection. RSV infection of 9HTE cells significantly upregulated the exp...
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