The Research Of Premature Chromosome Condensation As A Biological Dosimeter Induced By CA

Objective1To explore the best scheme of PCC （Premature chromosome condensation） in PBL （peripheral blood lymphocytes） induced by CalyculinA.2To explore the feasibility of CalyculinA induced premature chromosome condensation be used as a biodosmitry of low-dose ionizing radiation.,3There is a dose-rate effect between the chromosome aberrations and ionizing radiation in the low Linear Energy Transfer. In order to solve this problem, respectively observe dose effect relationship under different dose rate, and establish the dose-response curves and the corresponding mathematical model, So as to estimate exactly the biological dose of persons exposed to different dose rate.4To explore the feasibility of estimation of the irradiated fraction and dose using the Qpcc method in partial-body simulated exposures with6MV X-rays, premature chromosome condensation （PCC） induced by Calyculin A was analyzed.Methods16ml peripheral blood drawn from one healthy individual was inoculated on12culture bottles which were randomly divided into three groups. Lymphocytes were cultured for48hours in RPMI1640media, supplemented with20%（v/v） foetal call serum,1%（v/v） phytohemaglutinin,1%（v/v） HEPES, Colcemid was added24hours after cultures started and CalyculinA was added for the last two hours. Using factorial experiment design, to approach the two main factors influcing the chromosome condensation from the two levels and detect it by relative statistical methods. 2Peripheral blood was drawn from healthy adults and Anticoagulated with Heparin. a set of tubes was irradiated by X rays. The irradiation dose is0、0.1Gy、0.25Gy、0.5Gy、0.75Gy、1Gy separatly. After irradiation, The whole blood was added to each RPMI1640medium, All incubations were Cultured for up to48h at37℃in an atmosphere of5%CO₂in air. Colcemid was added24hours after cultures started and Calyculin A was added for the last2hours to induce pcc. The chromosome spreads were prepared, stained with Giemsa and observed under a microscope. The dose-response relationship of the total aberration, fragment, dicentric+centric ring（dic+r） in the irradiated lymphocytes was examined.1000G₁cells and1000G₂/M-PCC cells were analyzed in every dose by blind reading and the amount of chromosome aberration was counted and was analyzed by SPSS16.0. The experimental data was done curve fitting with three mathematical models supported by WHO and the fitted curve was determined by testing the significance of regression coefficient and the degree of curve fitting.312EP tubes（0.5ml） which seals6ml peripheral blood, drawn from healthy adults and anticoagulant with Heparin, were put in the socket in the solid water. The human peripheral blood was exposed by x ray in vitro with two different dose rates respectively, which is100MU/min and200MU/min. it was cultivated and harvested according to the conventional culture method. With blind reading, count the dicentric+ring chromosome aberration rate of the specimens. Through application SPSS16.0statistical software package to analyze the relation between chromosome aberration rate and irradiation, the dose-response curves and mathematical model were established in different dose rates.4Peripheral blood was drawn from healthy adults and Anticoagulated with Heparin. A set of tubes was irradiation by X rays. The irradiation dose included0、5、10、15、20、25Gy. After irradiation, The whole blood was cultured for52h with PHA and were forced to condense prematurely using50nM. The PCC index, PCC rings, and PCC fragments were scored for each dose point to arrive at the dose effect curve. At the same time, Partial-body accidental irradiations were simulated by in vitro irradiation of blood from healthy volunteer with8Gy6MV X-ray, the illuminated portion simulation local irradiation was20%,50%,80%, respectively.Results 1Different level of colchicine and CalyculinA training specimens:Lymphocyte activation,High concentration of CalyculinA in experimental groups was significantly higher than thats in the low groups in mitotic index, there was a statistical significance between each group in the whole experiment （P<0.05）.2The PCC was successfully induced in human peripheral blood lymphocytes with Calyculin A. The total aberration rate, fragment rate and dicentric+centric ring rate increases with the increase of irradiation dose, According to the statistical results of PCC aberration rate induced by different doses of X ray irradiation. Fitting the dose effect with SPSS16.0statistical software, the regression of equation, calculate the fit of equation （R²ï¼‰ and testing the significance of regression coefficients. Results show that in0-1.OGy dose range, these data were found to fit exactly with a quadratic polynomial dose response model, which could be described by the formula:y=a+bD+cD²3At same dose rate level, the aberration frequency increased with the absorpted dose, At same dose level the aberration frequency increased with dose rate, There is a pronounced dose-rate effect.4The results show that the PCC index decreases with the dose increases. The ring distribution of PCC fits poisson’s distribution in every dose. The ring rate and fragment ring of PCC increases with the exposure dose increases. The excess fragment of PCC, which can be used to estimate the partial exposure dose, in the damaged cells and exposure doses can be described by linear model.Conclusion1Lymphocytes was cultured for48hours, the mitotic index is optima when Colcemid with a final density0.04μg/ml was added24hours after cultures started and calyculinA with a final density60nM was added for the last two hours.2The premature chromosome condensation technique can be used as a technology to estimate the radiation dosage of low-dose ionizing radiation.3Considering the effect of dose-rate, approximate dose-rate dose-response curve should be chosen to estimate the absorbed dose in low linear energy transfer4Local exposure dose and portion can be more accurately estimated by Qpcc, so it can be used in measuring the level of local ionization damage by large doses.