RESUMO
PURPOSE: Eosinophils are cells of the granulocyte lineage that participate in host defence against parasitic disease and mediate allergic inflammation. In this study by using the combination of cytokines IL-3, IL-5 and GM-CSF, we explore the characterization of cultured eosinophils from CD34+ CBCs. METHODS: Mononuclear cells were isolated heparinized umbilical cord blood by Ficoll-paque (1.077 g/ml) density gradient centrifugation method. The CD34-bearing hematopoietic progenitor cells were collected by elution after their adhesion to a magnetic cell sepatation (MACS) column. The CD34+ cells were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and were cultured in the presence of IL-3, IL-5, and GM-CSF in a 6-well plate bottomed tissue culture plate at 37 degrees C for 7-28 days in humidified 5% CO2 and 95% air atmosphere. For the identification of cultured eosinophils Wright's and Giemsa staining, RT-PCR, Southern blotting and FACS analysis are used. RESULTS: We analyzed the cultured eosinophils Wright's and Giemsa staining, the total cell number of cells increased 50-fold by days 28 of culture. Also, using the sensitive RT-PCR technique, we monitered the appearance of mRNA transcrips of EPO. Identity of each RT-PCR product was confirms by southern blotting with independent gene-specific oligonucleotide probes and we found increasing of hybridization signals for EPO at 7th culture days. In addtion, we identified eosinophils in cultured CD34+ CBCs by flow cytometry. As s results, we succeeded in developing of pure eosinophils efficiently from CD34+ of CBCs in the presence of IL-3, IL-5 and GM-CSF. CONCLUSION: The in vitro growth of CD34+ CBCs may provide a useful system to study growth factor and stage-dependent adhesion molecule expression, as well as function on developing eosinophils.
