Producción CyT
Congreso
Autoría
Santos N
;
Freitas Rafael
;
ORONA, NADIA SOLEDAD
;
D.R. Tasat
;
Amaral A
Fecha
2010
Editorial y Lugar de Edición
Editora universitária UFPE
Resumen
Información suministrada por el agente en
SIGEVA
The aim of this study was to evaluate p21 protein expression levels of human peripheral blood lymphocytes by flow cytometry after exposure to gamma radiation. Peripheral blood samples from 5 individuals were apportioned in three aliquots: two were separately irradiated with gamma radiation (Cobalt-60) doses of 2 and 4 Gy, and one served as non-irradiated control. Mononuclear cells were isolated by Ficoll separation technique and resuspended in RPMI-1640 medium supplemented with 10 % fetal bovin...
The aim of this study was to evaluate p21 protein expression levels of human peripheral blood lymphocytes by flow cytometry after exposure to gamma radiation. Peripheral blood samples from 5 individuals were apportioned in three aliquots: two were separately irradiated with gamma radiation (Cobalt-60) doses of 2 and 4 Gy, and one served as non-irradiated control. Mononuclear cells were isolated by Ficoll separation technique and resuspended in RPMI-1640 medium supplemented with 10 % fetal bovine serum. Cell viability was checked by trypan blue exclusion and lymphocytes number was determined by counting in a manual hemocytometer (Neubauer chamber). Cell suspensions at density of 2 x 105 lymphocytes/well were deposited in 96-well plates and incubated, with and without phytohemagglutinin (PHA) (10 μg.mL-1), during 24, 48 and 72 hours, at 37 ºC and humidified atmosphere with 5 % CO2. Then, cells were harvested and labeled with phycoerythrin-conjugated anti-p21 (WAF1) monoclonal antibody. Flow cytometric acquisitions were performed on FACSCalibur equipment, by scoring of 50,000 events (cells) per sample, and FACS data were analyzed using FlowJo software. Statistical analysis was performed using analysis of variance (ANOVA), followed by Tukey’s posthoc test. Differences were considered statistically significant when p<0.05. The mean, minimal and maximal values of p21 expression levels from control group samples found were: quiescent – 24 h: 2.82±1.69; 0.75-4.82; 48 h: 5.12±5.99; 1.13-15.68; 72 h: 4.76±6.29; 1.97-15.97; proliferating – 24 h: 4.56±1.69; 2.9-7.4; 48 h: 10.02±10.46; 3.48-28.49; 72 h: 12.37±11.92; 3.89-32.11. Higher levels of p21 basal expression were observed in proliferating when compared to quiescent samples. For both stimulated and no-stimulated samples, lower p21 expression levels were observed for 24 hours-cultured samples, no differing statistically between 48 and 72 hours-cultured samples. No differences were observed between control and irradiated samples groups, to all culture time and conditions analyzed. However, the basal levels of p21 expression levels and the expression pattern in response to irradiation differ significantly among individuals, indicating that the response to DNA damage induced on the same irradiation conditions varies subject-to-subject, perhaps due to individual radiosensitivity. Analyzing group samples, alterations in response to irradiation were not observed; that differences only were identified through subject-to-subject evaluations. These results motivate further studies in order to investigate p21 protein expression levels as bioindicator of individual radiosensitivity.
Ver más
Ver menos
Palabras Clave
ymphocytesp21 proteinradiation