| Part I Quantitative assessment of DNA damage induced by CT radiation in human peripheral blood lymphocytes: compare with immunofluorescence and flow cytometryObjective To evaluate the accuracy of immunofluorescence and flow cytometry in the quantitative determination of DNA damage caused by CT radiation in the peripheral blood.Materials and Methods A total of 16 patients(mean age(23 ± 4.5)years)were selected from healthy volunteers.The peripheral venous blood of the volunteers was given 8ml and divided into 4 heparin anticoagulants by 2ml / tube and named for A,B,C and D tube.A tube is treated as the control tube without CT scan,But,B,C and D tube are treated under CT scan.Using the Siemens second generation dual source CT(Definition Flash,Siemens,Germany),set the scan parameters for each scan(voltage 120 kv,current 210 mAs,4D CareDose closed,scan FOV 250 mm × 250 mm,length 150 mm,rack rotation speed 300 ms / Rot,collimator width 0.65 mm,layer thickness 5mm),will contain 2ml venous blood heparin anticoagulant tube 4 / group level and placed in the CT scan bed,the B,C and D tube were once,twice and three times scanning.The Dose Length Product(DLP)of the CT examination was recorded.Within 5-10 minutes after CT scan,the lymphocytes were extracted,washing,fixed,rupture of membranes,closed,standard anti-a series of operations,dubbed 1ml suspension,and take 20 ml cell suspension evenly applied to non-slip offload Slides,dried after dyeing and DAPI sealing.The focus of γH2AX was observed and counted by fluorescence microscopy.Two patients were counted by double-blind method.The cells were homogeneous and the background was clear.Each sample counted 50 cells.The consistency of the two doctors was tested by kappa.The remaining suspension was sieved in 2 centrifuge tubes at 400 μl / tube,tube 1 as the background tube,tube 2 was added to the secondary antibody and incubated for 40 minutes and rinsed with 400 μl / tube suspension.The number of positive cells of γH2AX positive cells was counted and the percentage of positive cells per million cells was counted as Hist %.Results The coincidence Kappa values of immunodynamics γH2AX were 0.52,and the radiation dose DLP from B to D was(226.83 ± 13.22)mGy · cm,(448.5 ± 22.18)mGy · cm and(670.17 ± 31.92)mGy · cm.The γ H2 AX foci from A to D were(0.79 ± 0.351),(1.211 ± 0.509)and(1.750 ± 0.549)/ cells.The average amount of γH2AX measured by the flow cytometry(FCM)were(1.009 ± 1.961)%,(3.313 ± 3.778)%,(5.78 ± 5.046)% and(11.294 ± 10.793)%,respectively.The amount of γH2AX,which is measured by FCM and immunodynamics are all positively correlated with the DLP with r value of 0.63 and 0.74,respectively.Conclusion 1.Both inmmunofluorescence and flow cytometry can be used to quantitatively determine the biological effects of CT radiation.The amount of γH2AX measured by both is positively correlated with the number of radiation.2.Immunofluorescence method can be more intuitive to show the number and quality of γH2AX focus,but the steps are more cumbersome,susceptible to human factors interference,the consistency of the two observers is not high enough(kappa = 0.52).The FCM correlation between γH2AX and DLP,which represents radiation dose,was higher than that of immunofluorescence.Part II The effect of the iodine contrast agent on the biological effects of CT radiation(in vitro experiments)Objective: To evaluate the biological effects of the presence of iodine contrast agents in vitro on the DNA double-strand injury in CT Scan.Materials and methods: Peripheral venous blood was collected from 21 volunteers,12 ml / person,with an average of 2ml / tube,the blood was divided into 6 tubes.The tubes were signed by NO.1 to NO.6.NO.1 tube was treated with control tube,with 0.1ml normal saline added,NO.2 to NO.6 tube were added with 0.1ml of saline,0.05 ml iodine contrast agent and 0.05 ml physiological saline,0.1ml iodine contrast agent,0.1ml high concentration contrast agent,and 0.05 ml of contrast agent and 0.05 ml of saline.The NO.2 – NO.5 tubes are bundled into a 2 × 2 cube structure and flat on the CT scan bed.Using the Siemens second generation dual source CT(Definition Flash,Siemens,Germany),set the scan parameters for each scan(voltage 120 kv,current 210 mAs,close 4D CareDose,scan FOV 250 mm × 250 mm,length 150 mm,rack rotation speed 300 ms / Rot,collimator width 0.65 mm,layer thickness 5mm),on the 2nd,3 and 4,respectively,2 times the scan.The Dose Length Product(DLP)represents the radiation dose of the CT examination.After a CT scan,the peripheral blood lymphocytes were extracted by Ficoll stratification method,washed,fixed,ruptured,closed,labeled with a series of operations,and finally dubbed 800 μl suspension The tube A as a background tube,tube B added secondary antibody and incubation,40 minutes after the two tube washing,and dubbed 400 μl / tube suspension on the machine after the determination of the tube,A total of 10,000 cells were counted by flow cytometry.The number of γH2AX positive cells was counted and expressed as a percentage of positive cells per million cells.The ratio of positive γH2AX positive cells was Hist(% of%).Results The average radiation dose of NO.2 to NO.5 was(452.3 ± 26.5)mGy · cm,and the percentages of γH2AX from NO.1 tube to NO.6 tube were(0.45 ± 0.12)%,(4.40 ± 2.99)%,(6.38 ± 5.77)%,(7.28 ± 7.37)%,(4.36 ± 4.86)% and(4.89 ± 5.65)%,seperately.The percentage of γH2AX in tube NO.3 was higher than that of No.2.With the increase of iodine contrast agent concentration,NO.4 tube was higher than 3 tube,but when the iodine contrast agent concentration increased to 0.015 ml / tube,the γH2AX number decreased in the tube of NO.5.Administered alone with iodine contrast agent,the amount of γH2AX was significantly higher in NO.6 tube than that of the control tube.Conclusion 1.The presence of iodine contrast agents can "enlarge" the DNA injury in CT scan.This effect is associated with iodine contrast agent concentrations,but is reduced when beyond a certain concentration(24.42 mg I / ml in our trial).2.Iodine contrast agent itself can cause lymphocyte DNA damage alone.Part III The effect of the iodine contrast agent on the biological effects of CT radiation(in vivo experiments)Objective To evaluate the biological effects of the presence of iodine contrast agents in vivo on the DNA double-strand injury in CT Scan.Materials and method Sixty patients with suspected urinary tract disease who underwent CTU examination were randomly divided into control group and experimental group.Two groups of patients were divided into two times for CTU scan: the control group using conventional CTU examination(including plain,cortical,medulla and excretion of four),the first only the Department of urinary CT scan,scan Before and after 10 minutes of the extraction of elbow vein blood 2ml,were labeled A,B tube,three days after the addition of conventional CTU enhanced scan.The experimental group was divided into three groups: the first dose was injected with 50 ml of iodine contrast agent at the rate of 3 ml / s.After the injection was completed,the first injection was performed by fractional injection CTU.Patients were asked to sit in the examination bed,850 s after the same rate of re-injection of 40 ml of contrast agent,and then 30 s after the start of the real-excretion scan)were scanned before the iodine contrast agent 10 minutes after injection and 10 minutes after the scan Extraction of elbow venous blood,and were labeled as A1,C and B1.The peripheral blood lymphocytes were isolated and fixed by 4% paraformaldehyde.Antibody was incubated.Finally,the content of γH2AX in each tube was quantitatively determined by flow cytometry.The changes of γH2AX content in AB and A1-B1 tubes were analyzed to evaluate the effect of iodine contrast agent on the DNA of human peripheral blood lymphocytes induced by CT radiation.The changes of γH2AX in the C tube relative to the A1 tube were evaluated and the iodine contrast agent The biological effects of DNA on lymphocytes.Results The levels of γH2AX in the control group and the experimental group were(19.204 ± 5.942)% and(20.217 ± 7.498)%,respectively,the difference was not statistically significant(t = 0.11,P = 0.94);after scanning were(31.219 ± 7.780(12.015 ± 2.119)% and(19.043 ± 3.452)%,respectively.The differences were significant(12.015 ± 2.119)% and(19.043 ± 3.452)%,respectively.The difference was statistically significant(t =-3.09,P = 0.00)(T =-2.58,P = 0.00).The number of γH2AX in the experimental group was 58.49% higher than that in the control group.The content of γH2AX in the peripheral blood of the control group before and after the injection of iodine(10 minutes)20.217 ± 7.498)and(26.100 ± 10.743)%,the difference was statistically significant(t =-2.12,P = 0.00),and γH2AX increased by 29.08% after injection of contrast agent.Conclusion 1.Iodine contrast agent in the blood environment can increase the number of DNA damage caused by CT examination by 58.49%;2.Iodine contrast agent itself can cause peripheral blood lymphocytes DNA damage alone.Part IV Self-repair of induced DNA Damage in Human Peripheral Venous Lymphocytes Cells :Comparation of Iodine Contrast Agent and CT RadiationObjective: To evaluate the Self-repair difference of human peripheral blood lymphocytes induced by X-ray radiation and iodine contrast agent.Materials and methods: Health volunteers were selected as the study subjects.A total of 10 patients(mean age 42 years)were selected.Each volunteer was extract 16 ml of peripheral venous blood and were divided into 8 tubes with 2 ml / tube.Eight tubess were divided into A and B groups with 4 tubes each group.A1-A4 group were added with 50 μl iodine contrast agent(350mgI / ml)and B1-B4 tubes were added with 50 μl physiological saline and scan once each tube.Using the Siemens second generation dual source CT(Definition Flash,Siemens,Germany),set the scan parameters for each scan(voltage 120 kv,current 210 mAs,close 4D CareDose,scan FOV 250 mm × 250 mm,length 150 mm,rack rotation speed 300 ms / Rot,the width of the collimator was 0.65 mm and the layer thickness was 5 mm),and 4 ml of the heparin anticoagulant tube containing 2 ml of venous blood was placed side by side in the CT scan bed and 1 scan scan of the B1-B4 tube.The Dose Length Product(DLP)represents the radiation dose of the CT examination.After the injection of iodine contrast agent or after the end of the scan,the blood samples were stored in a sterile incubator at 37 ° C to maintain activity.The blood was treated at 30 min,2 h,4 h and 8 h after added with iodine contrast agent or performed with CT scan.The peripheral blood lymphocytes were extracted by Ficoll stratification method,washed and fixed,ruptured,ruptured,closed,and labeled with a series of operations.Finally,a suspension of 800 μl was prepared and treated with 400 μl in the two centrifuge tubes,tube A as the background tube and tube B added secondary antibody and incubation.Wash the tubes after 40 minutes and start counting in flow cytometry,counting 10000 cells automatic.the final proportion of γH2AX positive cells were recorded by Hist(% of the test tube%).Results The average radiation dose of B1-B4 was(234.25 ± 14.11)mGy · cm.The γ H2 AX in tube A1-A4 were(38.07 ± 21.29)%,(43.50 ± 25.52)%,(33.60 ± 10.46)% and(28.31 ± 8.69)% respectively at the time of 30 min,2h,4h and 8h after been treated.The γ H2 AX in tube B1-B4 were(34.29 ± 19.92)%,(49.27 ± 16.72)%,(14.21 ± 10.70)% and(7.82 ± 3.49)%,respectively.The γ H2 AX reached the maximum amount at the time of 2h in both groups,but the amount of γH2AX in group B was significantly lower than that in group A after 4 hours.Conclusion The body has the ability of self-repair after the DNA double-chain injury by X-ray or iodine contrast agent.The number of γ H2 AX reached the peak in the second hour after been induced.But the number of γ H2 AX is higher than that of X-ray induced group. |
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