12th Congress of the European Hematology ... - Haematologica

1466
ROS GENERATION AND APOPTOSIS IN HUMAN UMBILICAL STEM CELLS BY LOW DOSE
RADIATION. PROTECTIVE EFFECT OF IGF1
K. Floratou, 1 S. Taka, 2 G. Antonakis, 3 D. Kardamakis, 4 P. Matsouka 5
1 University of Patras, Medical School, PATRAS; 2 Laboratory of Hematology,
PATRAS; 3 Department of Obstetrics and Gynecology, PATRAS; 4 Laboratory of
Radiobiology, PATRAS; 5 Laboratory of Haematology, PATRAS, Greece
Backrgound. CD34 cells are sensitive to radiation. Apoptosis may be
triggered by alterations in energy production system, translated in ROS
generation by the mitochondria of irradiated cells. Aims. The aim of our
study was to investigate the sensitivity of CD34 cells to low dose radiation
and the possible inhibitory/protective role of IGF1 on free radical
production by the mitochondria of the irradiated human CD34 umbilical
stem cells. Methods. Cord blood was collected from normal full-term
deliveries and CD34 cells were isolated using MACS system. The purity
of isolated cells was evaluated by flow cytometry using a FITC conjugated
monoclonal anti-CD34 antibody. CD34 cells were diluted to
25×10 3 cells/mL and cultured with IMDM supplement with 20% BIT.
Cells were irradiated with 0, 1, 2 and 5Gy and IGF1 was added to the
culture (100 ng/mL) 30 min prior to radiation. Apoptosis was evaluated
6 and 24h after irradiation by flow cytometry using Annexin-V/PI kit.
ROS generation was evaluated by FACS analysis 30min and 24h after
radiation using a) Hydroethidine staining of cells for O2. - and b)
chloromethyl-H2DCFDA staining (molecular probe) for H2O2. For statistical
analysis we used two paired t-test. Results. 1. Thirty minutes after
radiation with 1, 2 and 5Gy, the percentage of irradiated cells that generated
O2.- increased to 5, 11 and 30%, compared to the unirradiated cells
innate production. At the same time points, H2O2 increased 4, 8 and
20%, respectively. 2. Twenty four hours after radiation, the irradiated
cells continued to generate O2.- relatively to radiation dose 1, 2 and 5Gy,
in percentage 11.2, 18.6 and 26% compared to innate production of unirradiated
cells, but the fraction of H2O2 producing cells was not affected.
3. The addition of IGF-1 inhibited the ROS production by the irradiated
cells significantly at higher doses, at both early and late time
points. IGF-1 inhibited percentage of irradiated cells that generated O2.-
11% and 20% in 30min after 2 and 5Gy, respectively and 8.5% and 8%
in 24h. The fraction of H2O2 producing cells was inhibited by IGF-1 in
30 min 6% and 13% after 2 and 5Gy, respectively. 4. Six hours after
radiation with 1, 2 and 5Gy, apoptosis was increased 31.6%, 37.35% and
42.95% respectively (p