| Ionizing radiation is the general name that all can cause the material to be ionized. Nowadays,along with the great development of nuclear techniques, ionizing radiation technology has been widely used in the field of medicine, industry, agriculture, energy, geology, military science as well as the daily life and so on. It brings human beings with not only remarkable social effects and economical interests, but also radiation injuries or what are even worse, disasters, given different purpose of use or wrong preventive measures. Hence, fast and accurate detections of dosage and risk of a sufferer on a very early stage are vital for treatment. Furthermore, to estimate the radiation dosage is the key point in experimental studies. What we already have, which are some certain biological dosage indicators and methodologies, are practical to some extent, but far from satisfactory one. In this work, I am trying to find a new biomarker, which can help us in fast and accurate estimations on radiation sufferer, to direct the clinical treatment.ObjectivePrevious reports about the possible changes of MN-RET in the peripheral blood of mice detectded by flow cytometer, mostly refered to chemical mutagenesis. Studies on the dose-effect of MN-RET induced by ionizing radiation in the peripheral blood of mice detected by flow cytometer were rarely found. Flow cytometer was used to detect the possible changes of MN-RET in the peripheral blood of mice irradiated by X-ray in this study, aiming to confirm the possibility of using it as a radiation biomarker. At the same time, if ionizing radiation can cause CCND1 gene overexpression and this overexpression has dose-effect relationship were studied in this experiment. This results will establish a experimental foundation for its application to estimate radiation dosage. Methods1. Detection of micronuclei in polychromatic erythrocyte from bone marrowFifty-four ICR mice were divided randomly into 24h group, 48h group and 72h group .Each group was then divided into six subgroup and per subgroup contained three mice. The single whole-body radiation dose was 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 5Gy respectively. MNPCE in the bilateral thighbone marrow of mice irradiated by X-ray was detected using the double-blind method after mehanol fixation and Giemsa dyeing.Two-thousand PCE were observed in each mouse. The rate of MNPCE was expressed by permillage.2. Detection of micronuclei in reticulocytes from peripheral BloodObtaining the blood from eyeball, 50μl CD-71 antibody, 1μl RNA enzyme, and 50μl the PI staining were added to EP tube containing 70μl eyeball-blood dealed with heparin which was fixed for 24h by 75% hypothermia mehanol. Then MN in RET of peripheral blood was detected by Flow Cytometer. Ten-thousand RET was collected in each mouse.3. Detection of CCND1 gene expressionThirty-six ICR mice were divided randomly into 4h group and 24h group . Each group was then divided into six subgroup and per subgroup contained three mice. The single whole-body radiation dose was 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 6Gy respectively. Total RNA from thymus of mice irradiated by X-ray was isolated using RNAiso Plus and reverse-transcribed. The generated cDNAs was subjected to real-time PCR using the primer pairs of CCND1 .Oligonucleotide primers for PCR were designed using Primer Express software .Results1. Changes of micronuclear rates in polychromeatic erythrocyte of mice irradiated by X-rayMicroscopic assay results showed that with the radiation dosage increasing from 0Gy to 5Gy, rates of MNPCE elevated from 2.000‰, 3.000‰, 2.000‰to 35.333‰, 23.333‰, 21.334‰respectactively in 24h, 48h and 72h. In 0~5Gy dosage range, rates of MNPCE increased along with the increasing of dosage and there was a good dose-effect relationship (24h Y=3.708+10.447X-0.84X2 , r=0.996 , 48h Y=3.358+5.9X-0.41X2,r=0.996,72h Y=2.157+6.095X-0.457X2 r=0.999) in 24h, 48h and 72h after X-ray radiations. But, rates of MNPCE decreased in 48h comparing with 24h, also in 72h comparing with 48h.So it was best to observe MNPCE in 24h.2. Changes of micronuclear rates in reticulocytes from mice peripheral blood irradiated by X-rayMicronuclei in reticulocytes from mice peripheral blood was detected in 24h, 48h and 72h. As the radiation dosage increased from 0Gy to 5Gy, micronuclear rates increased from 3.10‰,3.13‰,3.07‰to 43.70‰,33.43‰,29.73‰respectactively, and it showed a significant dose–effect relationship (24h Y=4.258+10.605X-0.557X2 r=0.998,48h Y=4.163+5.472X+0.075X2 r=0.997,72h Y=4.461+2.899X+0.397X2 r=0.991), especially in 24h , which was nearly a straight line.3. The comparision of micronuclear rates in reticulocytes from peripheral blood and micronuclear rates in polychrometic erythrocytes from bone marrow of mice irradiated by X-rayNot only were micronuclear rates in reticulocytes from peripheral Blood and micronuclear rates in polychrometic erythrocytes from bone marrow in 24h group ,48h group and 72h group raised ,but also was there nice dose-response realationship and good correlation between them after X-ray irradiation(r changed from 0.955 to 0.989 and p is less than 0.01). Micronuclear rates in reticulocytes from peripheral blood detected by flow cytometer was higher than micronuclear rates in polychrometic erythrocytes from bone marrow in the same experiment condition.4. Changes of CCND1 gene expression in mice irradiated by X-rayIt showed that when giving 0.5Gy X-ray irradiation CCND1 gene expression have relative decline but little difference and no statistically significant . As the radiation dosage increased from 1Gy to 4Gy, CCND1 gene expression in 4h and 24h increased along with the dosage increasing. The expression peaked in 4h when giving 4 or 6Gy X-ray radiations, it was 2.35 times comparing with control group. CCND1 gene expression also peaked in 24h when giving 4Gy X-ray radiation, it was 3.17 times comparing with control group.But it was lower comparing 6Gy with 4Gy, and was only 1.6 times compared with control group(4h Y=0.793+00.597X-0.555X2 r=0.997,24h Y=0.474+1.904X-1.445X2 r=0.854).Conclusion1. Ionizing radiations can cause an increase of micronuclear rates in PCE from bone marrow of mice, and this increase has a significant dose–effect relationship.2. Ionizing radiations can also cause an increase of micronuclear rates in RET from the peripheral blood of mice, and this increase has a significant dose–effect relationship. MN in RET detected by employing flow cytometer (FCM) can be regarded as a biomarker of ionizing radiation, however the actual effort is to be researched further before its application on human body.3. In certain dose range and certain points of testing time,ionizing radiation can cause an over expression of CCND1 gene, and has some certain dose–effect relationship; CCND1gene expression reflected the radiation dosage to some extent, though it is not for sure to be used as a biomarker.
|
PDF Full Text Request