Studies On The Expression Of DNA Damage Response Molecules As Ionizing Radiation Biomarkers

With the increasingly widespread application of nuclear energy, nuclear radiation accidents are inevitable, more and more countries attach importance to the safety of nuclear applications and have set up and improved the emergency mechanism responded to nuclear radiation accident. Accurately estimating the dose received is the primary step for treating wounded graded, for the public, we need to find biological samples which are sensitive, reliable and rapid-detected as markers of irradiation to determine the severity of injury, and these researches have also become a hot spot .γ-H2AX and p21 protein are radiation-related proteins, p21 gene is a radiation-related gene. They all participate in radiation-induced DNA DSB repair, play an important role in cell cycle regulation and maintenance of genome stability, and are considered to be new irradiation biomarkers. In this study, we detected the expression ofγ-H2AX, p21 protein and gene after different time and dose of X-ray irradiation through FCM, then summed up their changes, and provided a scientific basis forγ-H2AX, p21 protein and p21 gene as biomarkers of irradiation.1. Effects of irradiation on the expression ofγ-H2AXThe expressions ofγ-H2AX in Jurkat cell lines were detected by flow cytometry after treated with 2 Gy X-ray at 0.5 h, 1h, 4h, 8h, 16h and 24h. The results showed that there was a remarkable increase at 0.5h, up to the peak at 1h, followed by a time-dependent decrease, and down to no significant difference with the control at 24 h.The expressions ofγ-H2AX in Jurkat cell lines were detected after treated with 0.5, 1, 2, 4, 6 Gy X-rays at 1h. The results showed that the expressions ofγ-H2AX were separately 1.6 times, 5.8 times, 8.6 times, 11.6 times and 7.8 times as much as the control, showed a dose-dependent increase in the range of 0.5～4 Gy, and dropped at the does of 6 Gy. Detected at 8 h after exposed to X-rays with those doses above, the express levels ofγ-H2AX were separately 1.8 times, 3.6 times, 4.7 times, 7.6 times and 5.8 times as much as the control, and in the range of 0～4 Gy, the expression ofγ-H2AX increased linearly with the dose increased, showing a clear dose-dependent manner. Overall,γ-H2AX expressions in Jurkat cell lines were at high levels during 1 h～8 h, and the express level of 8 h was lower than 1 h. It demonstrated thatγ-H2AX was sensitive to radiation, and its expression had a reasonable dose-effect relationship in a certain range of dose.2. Effects of irradiation on the expression of p21 proteinThe expressions of p21 protein in Jurkat cell lines were detected by flow cytometry after treated with 2 Gy X-ray at 4h, 8h, 16h and 24 h. The results showed that the expressions of p21 protein increased time-dependent, reached the peak at 24h, then decreased at 48 h, but still significantly higher than the control.in Jurkat cell lines were detected after treated with 0.5,1,2,4,6 Gy X-rays at. The results showed that the expressions of p21 protein increased significantly with the dose increased in the range of 0.5～4 Gy at both 12 h and 24 h, showing certain linear relationships, and decreased at the does of 6 Gy. Overall, the expressions of p21 protein were higher at 24 h than at 12 h. It implicated that radiation can induce the increase of p21 protein expression, and had a dose-dependant relationship in certain range of dose.3. Effects of irradiation on the expression of p21 geneThe relative expressions of p21 gene in mouse thymus and spleen cells were detected by Real-Time PCR after treated with 0.5, 1, 2, 4, 6 Gy X-rays at 4 h and 24 h. The results showed that the relative expressions of p21 gene showed dose-dependent increase in ICR mouse thymus cells, and there was not much difference between the two time points. Because of the complexity of the spleen cells, the expressions of p21 gene were higher at 4 h than at 24 h, but still showed dose-dependent increases compared with the control. The results demonstrated that radiation increased p21 gene expression, showing good linear relationships, and the peak of p21 gene expression appeared early than p21 protein's.4.γ-H2AX,p21 protein and p21 gene as irradiation biomarkersBy making linear fits, it was proven that the dose-effect results ofγ-H2AX,p21 protein and p21 gene all had reasonable linear relationships at their own time points studied, and reached better r and P values, as well as the requests of irradiation biomarkers.