The Studies On Dose-effect Relationship Of MN-RET From Peripheral Blood In Mice Induced By Ionizing Radiation

Along with the great development of scientific technology, ionizing radiation technology has been widely used in the field of medicine, industry, agriculture, energy, geology and so on. It brings human beings with not only remarkable economical interests, but also radiation injuries or what are even worse, disasters under special circumstances. Fast and accurate detections of dosage and risk of a sufferer on a very early stage are vital for early diagnosis, treatment and prognosis guidance after judge and distant effect evaluation.Hence. to find fast.accurate and high-throughput radiation biodosimeter is the key point in the field of radiation injury research. ObjectiveDose-response relationship of MN-RET induced by X-ray radiation of different dose rate in the peripheral blood of ICR mice detected by flow cytometer were researched. Dose-response relationship of MN-PCE induced by X-ray radiation of different dose rate in the bone marrow of mice observed by microscope under the same conditions to verify its objectivity and authenticity to establish a experimental foundation for its application to estimate radiation dosage.At the same time flow cytometer was used to detect the apoptosis rate of erythroblast from bone marrow in mice induced by 2Gy X rays of different dose rate, discussing the mechanism of micronucleus formation from the cells apoptosis.Methods 1. Detection of MN-RET from peripheral blood in mice induced by X ray irradiation of different dose rateNinety-six ICR mice were divided randomly into0.5Gy/min group.1.0Gy/min group,2.0Gy/min group and 3.5Gy/min group.Each group was then divided into six subgroup such as 0Gy.1.0Gy,2.0Gy.3.0Gy.4.0Gy and 5.0Gy and per subgroup contained four mice. The blood from eyeball in mice was collected at 24h after single whole-body irradiated and fixed over 24h in-70℃refrigerator.CD-71 antibody. RNA enzyme and the PI staining were added to the blood. Twenty-thousand RET were collected by FACScan Flow Cytometer for micronuclear analysis.2. Detection of MN-PCE from bone marrow in mice induced by X ray irradiation of different dose rateMice were killed by cervical dislocation after obtaining the blood from eyeball. MN-PCE in the bilateral thighbone marrow of mice irradiated by X-ray of different dose rate were scored with manual microscopy after mehanol fixation and Giemsa dyeing.Two-thousand PCE were observed in each mouse. The rate of MN-PCE was expressed by permillage.3. Detection of apoptosis rate of erythroblast from bone marrow in mice induced by different dose rate of 2GyX ray irradiationTwenty ICR mice were divided randomly into 0Gy/min group.0.5Gy/min group,1.0Gy/min group,2.0Gy/min group and 3.5Gy/min group. The first group was control group and the following 4 groups were all irradiated by 2Gy. After 24h irradiation, the mice were killed by cervical dislocation, then bilateral bone marrow was washed with 5ml cold PBS buffer and filtered with 300 mesh filter and centrifuged, lastly it was made into single cell suspension which containded 1.0×106 cell per mil.CD71 antibodies, Buffdr buffer. Hochest33342 solution and PI solution were added respectively to cell suspension. Ten-thousand CD71-positive cells were collected by FACScan Flow Cytometer for cell apoptosis analysis.Results 1. Dose-effect relationship of MN-RET from peripheral blood in mice induced by X ray irradiation of different dose rateMN-RET rates all increased with dose in the range of 0-5 Gy with X-ray radiation of different dose rate. The dose-response relationship all can be fit with linear equations, but slope between different dose rate groups is distinct.Statistical analysis showed that the regression coefficients among 0.5Gy/min group vs 1.OGy/min and 2.OGy/min(to.5Gy/min vs 1Gy/min= 6.881. P<0.05; to.5Gy/min vs 2Gymin= 4.767. P<0.05). 1.OGy/min group vs 2.0Gy/min and 3.5Gy/min(t1Gy/min vs 2Gymin 2.612. P<0.05; t1Gv/min vs 3.5Gy/min= 4.896. P<0.05).2.0Gy/min group vs 3.5Gy/min(t2Gymin vs 3.5Gy/min= 2.756. P<0.05) have a statistically significant difference. The regression coefficients between 0.5Gy/min group vs 3.5Gy/min have no significant difference.2 Dose-effect relationship of MN-PCE from bone marrow in mice induced by X ray irradiation of different dose rateMN-PCE rates all increased with dose in the range of 0-5 Gy with X-ray radiation of different dose rate.The dose-response relationship all can be fit with linear equations, but slope between different dose rate groups is distinct.Statistical analysis showed that the regression coefficients among 0.5Gy/min group vs l.OGy/min group,2.0Gy/min group and 3.5Gy/min (t0.5Gy/min vs 1Gy/min= 7.63.P<0.05; t0.5Gy/min vs 2Gymin= 6.47, P<0.05; t0.5Gy/min vs 3.5Gy/min= 2.51, P<0.05):1.OGy/min group vs 2.0Gy/min and 3.5Gy/min (tiGy/min vs 2Gy/min= 2.32, P<0.05; t1Gy/min vs 3.5Gy/min= 5.05. P<0.05);2.0Gy/min group vs 3.5Gy/min (t2Gymin vs 3.5Gy/min=3.56.P<0.05) have a statistically significant difference.3. Relationship of MN-RET from peripheral blood detected by FCMand MN-PCE from bone marrow detected by manual microscopyResults from FCM and manual microscopy both showed a good dose-effect relationship between MN and dose, but results from FCM were slightly higher than artificial microscopy's, statistical analysis showed that there was no significant difference b etween results from two methods (t0.5Gv/min= 1-724. P> 0.05; t1.0Gv/min= 2.157, P> 0.05; t2.0Gy/min=1-288. P> 0.05; t3.5Gv/min= 1-333. P> 0.05). and there were good correlations between them (r0.05ay/min= 0.987. P<0.05:r1.0Gy/min= 0.993. P <0.05; r2.0Gv/min= 0.992. P<0.05; r3.5Gv/min= 0.989. P<0.05).4. Changes of apoptosis rate of erythroblast from bone marrow in mice induced by 2GyX ray of different dose rateAfter mice were irradiated by 2Gy X ray of different dose rate.apoptosis rates of erythroblast from bone marrow have relative rise and statistically significant differences compared with the control group(P<0.05). As the radiation dosage increased.there were significantly differences among the radiation groups(P<0.05).Conclusion1. MN-RET rates all increased with dose in the range of 0-5 Gy with X-ray radiation of different dose rate, the dose-response relationship all can be fit with linear equations,, but slope between different dose rate groups is distinct.2. MN-PCE rates all increased with dose in the range of 0-5 Gy with X-ray radiation of different dose rate, the dose-response relationship all can be fit with linear equations,, but slope between different dose rate groups is distinct.3. There was no significant difference between results from two methods and there were good correlations between them. MN in RET detected by employing flow cytometer (FCM) can be regarded as radiation biodosimeter, however the actual effort is to be researched further before its application on human body.4.Apoptosis and micronuclei formation are two forms of damage of ionizing radiation, they coexist, micronuclei formation was main in lower dose rate irradiation, but apoptosis was main in higher dose rate irradiation.