| The wide use and continual expansion of atomic energy industry and its related technologies have caused hazards to human beings when it brings benefit. As an important aspect of the radiation hazards evaluation, At present, chromosome aberration analysis is the preferred approach applying to biological dose assessment. However, this approach also has obvious defect that cannot be early, high-throughput estimating the dose, and is too complex and needs too much time. Therefore, establishment of the methods on early, quick and high throughput biological dose evaluation using modern biological technology and advanced methodology has become the research hotspot in the field of radiation biology.GADD45 gene is a member of the Growth arrest and DNA damage inducible genes family, and is adjusted by a variety of factors both inside and outside cells at the transcriptional level, post-transcriptional level, translation level and other levels. GADD45A is the gene that can be strongly induced by ionizing radiation and react on gene expression. This gene expression change as a biological dosimeter has become the focus in recent years.Nm23 gene can suppress the cancer metastasis. Related studies have shown that ionizing radiation can exert certain effects on the expression of nm23-H1 gene, but most of these researches focused on the study of tumor cells. That whether the nm23-H1 gene expression changes in tumor cells and healthy human peripheral blood cells after ionizing radiation are coincident has not been reported, so have the studies about the effect of ionizing radiation on expression of nm23-H1 in human peripheral blood cells.ObjectiveThe topic studied the expression changes of GADD45A and nm23-H1 genes in human peripheral whole blood cells exposed to X-ray irradiation in vitro using the quick, accurate and high-throughput Real Time quantitative RT-PCR method, and analyzed the dose-response relationship and time-course change, so as to provide a method and experimental basis for finding a new specific biomarker of radiation and dose estimation system.Meterials and Methods1 Sample collection, irradiation and cultureIn the case of informed consent, human peripheral whole blood from four healthy individuals was collected by venipuncture into vacutainers containing EDTA-K2, then exposed to X-rays with the doses between 0 and 5 Gy, respectively. The samples were cultured in 1640 medium for 0,6,12,24 h when cells were harvested.2 White blood cells separation and extraction of total cellular RNAErythrocyte lysis solution disrupts the red blood cells, and the white blood cells were washed by phosphate buffer saline. Then the total RNA were extracted using Trizol method.3 cDNA synthesisThe total RNA were transcripted into cDNA using Random Primers and M-MLV reverse transcriptase. The cDNA were stored at-20℃and spare. 4 Real Time RT-PCR18S rRNA as a reference gene, Real Time quantitative RT-PCR was used to detect the relative expression levels of GADD45A and nm23-Hl gene with different doses. Then observe the dose-response relationship and time-course change.5 Statistical analysis and processingData of the experiment were processed by SPSS software 12.0 with one-way anova. Size of the test wasα=0.05.Results1 The dose-effect and time-course relationship of GADD45A geneExpression levels of GADD45A gene after X-rays irradiation were significant difference between every group at different time points (F=10.497,8.753,31.874, P<0.05). The levels of GADD45A gene expression increased gradually with the doses from 0 to 5 Gy at 12 h, and the dose-response relationship was better. The equation of dose-effect curve at 12 h is Y=10-0.878+0.118X-2, R2= 0.605. The levels also increased from 2 to 4 Gy at 24 h. GADD45A gene expression levels constantly increased over time after irradiation (F= 76.431,23.771,13.797,99.805,18.834, P< 0.01).2 The dose-effect and time-course relationship of nm-23-H1 geneThe levels of nm-23-H1 gene expression increased gradually with the doses from 1 to 3 Gy at 6 h (F= 15.064, P< 0.05), and from 1 to 5 Gy at 12 h (F= 3.435, P < 0.05) after X-ray irradiation, and the equation of dose-effect curve at 12 h is Y=10-0.441+0.085X, R2=0.309. However, gene expression levels of 0 and 24 h were no significant difference between various groups (F=0.478,0.444, P> 0.05). The time effect curve showed that the expression of nm23-H1 gene decreased with the doses of 0,4 and 5 Gy after irradiation (F= 8.636,4.112,5.411, P< 0.05). Meanwhile, the expression of nm23-H1 gene was the lowest at 24 h with 2 and 3 Gy irradiation (F =12.517,6.622, P<0.05).Conclusions1 The expression changes of GADD45A and nm23-H1 gene in human peripheral blood cells after irradiation in vitro increased gradually with the increase of radiation doses in different dose ranges, and showed the dose-effect relationship.2 The dose-effect relationship of GADD45A gene expression change induced by ionizing radiation is superior to that of nm23-H1 gene.
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