The Study Of HBD-2Antimicrobial Immunodefensive Function In Vitro

Objective:To construct a primary culture model of human nasal mucosa epithelial cells in vitro; evaluation the proteins antimicrobial activity of the cell model products which are transfected by hBD-2gene; to provide a basis for further study of hBD-2immunodefensive function in vivo.Methods:1.Collect the inferior turbinate mucosa, enzyme digestion combined with tissue block method to culture the nasal mucosa epithelial cells in vitro, and identification the cell by immunohistochemistry;2. cationic liposome Lipofectamine2000mediated pIRES2-Zsgreen1-hBD2recombination plasmid be uesed to transfect nasal mucosa epithelial cells, detect the expression of hBD-2by fluorescence microscope and quantitative RT-PCR;3. The cell culture supernatant were collected for evaluation the antimicrobial activity of the rhinosinusitis common bacteria by punch method.Results:1.the primary human nasal mucosa epithelial cells cultured in vitro were identified as epithelial cells by cytokeratin AE1/AE2,which positive rate was approximately93.1%;2. The green fluorescence could be showed in the transfected plasmid cells by fluorescence microscope, and the transfection efficiency of the recombinant hBD-2cell was approximately35%. The expression of hBD-2mRNA in the transfected hBD-2cell could be detected increased by the quantitative RT-PCR3.Cell culture supernatant from hBD-2transfected produced bacteriostatic ring on Escherichia coli, Staphylococcus aureus,Pseudomonas aeruginosa and Streptococcus pneumoniae.Conclusion:1.enzyme digestion combined with tissue block method culture the nasal mucosa epithelial cells have a high rate of separation and speeds of propagation;2.The cell which Liposome mediated hBD-2transfection can effectively express hBD-2;3.The hBD-2transfected nasal mucosa epithelial cell could secrete effective antimicrobial peptide and express antimicrobial activity in vitro.9figures,1table,33references.