The Study On The Radiosensitivity Of AT Cells

The radiosensitivity is one of the main correlative factors with the organism to affect the biological effects. The difference of sensitivity to ionizing radiation between various species, tissues and cells, as well as the biological macromolecules is very significant, namely, in the same condition, damage or hormesis effects on different organisms, tissues, cells or biological macromolecules caused by the same dose of the identical ray may be distinct. It is clear that the radiosensitivity of the cell is mainly related to the concentration of DNA in the cells, the kind and the biological structure and the cell cycle phase of the cell, the exterior and interior environment for the cell to grow in, the rate for the cell to proliferate, the capacity for the cell to repair DNA damage and scavenge free radicals produced by itself or other cells. With the development of clinic radiation therapy of the tumors and the subject for radiation protection, the studys on the radiosensitivity of the cell are gradually becoming a focus in the field of radiobiology in the latest 20 years.Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive cerebellular degeneration, radiation hypersensitivity, immunodeficiency, genomic instability, premature aging and predisposition to cancer. Cultured AT cells lack radiation-induced cell cycle checkpoints and express cellular defects that correlate with the clinical phenotype. AT is caused by functional inactivation of the ATM gene on 11q22-23. It is assumed that ATM is an early key component ofa signal transduction network that mediates cellular responses to certain types of DNA damage. Based on sequence homology, ATM belongs to a family of large eukaryotic proteins that regulate cellular responses to DNA damage, including DNA repair, genetic recombination, apoptosis and cell cycle checkpoints.Objective: Compared with those of GM fibroblasts, the abilities for AT fibroblast to produce superoxide anion free radical (O2")and incorporate 3H-TdR and repair DNA double strands breakages, the ratio of chromosomal aberrations and apoptotic cell, the ratio of GO/G1, S, G 2/M during cell cycle of AT fibroblast after exposure to 60Co Y rays were detected, in order to study the effects of ionizing radiation on the proliferation, the distribution of cell cycle, apoptosis, chromosomal aberrations and DNA double strands breakages and repair of AT fibroblasts, and the mechanism of the radiosensitivity of AT fibroblasts was primarily explored.Methods: (1) Cell Culture: All fibroblasts were grown in DMEM or RPMI1640 medium containing 15%FBS and lOOU/ml penicillin and streptomycin at 37 Cin 5% CO2. subculture per 3d; (2) Irradiation: cells were irradiated to 60Co Y source at a dose rate of IGy/min; (3) Chromosomal aberration analyses: samples were prepared by routine method, 400-1200 metaphases were analyzed for microscopically detectable chromosome type aberrations; (4) Cell cycle distribution: According to the concentration of DNA, the ratio of GO/G1, S, G 2/M was determined by FACS;(5) Apoptosis: DNA was eluted with phenol-chloroform approach and electrophoresized to separate DNA fragments and photographed, cells were treated with Annexin V-FITC Apoptosis Detection Kit I and the percentage of apoptotic cells weredetermined using FACS.(6) DNA double strands breakages and repair: pulsed field gel electrophoresis (PFGE) were used to detect the distribution of DNA fragments in a CHEF MAPPER?system at the very moment or at different time after 60Co Y ray irradiation. (7) 3H-TdR incorporation: trypsinizing the anchored fibroblasts during logarithmic growth phase with 0.25% trypsin and sampling by filter method and determining the CPM value of fibroblasts by liquid scintillation counter; (8) [O2"] determination: fibroblasts were culture in RPMI11640 medium without phenol -sulfonphthalein, cytochrome C was added into the medium at 5h before harvest, supernatant was collected by centrifugation, the optical density value was determined by type-722 photometer and...