【作者】 李强；

【导师】 胡春洪；

【作者基本信息】 苏州大学 ， 影像医学与核医学（专业学位）， 2017， 博士

【摘要】 第一部分免疫荧光和流式细胞法定量评估CT辐射致人体外周血淋巴细胞DNA损伤的对照研究目的评估免疫荧光和流式细胞仪两种方法定量测定CT辐射致人体外周静脉血DNA损伤的准确性。材料与方法选择健康志愿者为研究对象,共16例,平均年龄（23±4.5）岁,抽取志愿者的外周静脉血8ml,以2ml/管平均分入4个肝素抗凝管中,分别命名为A、B、C和D管。A管为对照管,不进行CT扫描,B、C和D管进行CT扫描。使用西门子第二代双源CT（Definition Flash,Siemens,德国）,设定每次扫描参数一致（电压120kv,电流210mAs,关闭4D CareDose,扫描FOV 250mm×250mm,长度150 mm,机架旋转速度300ms/rot,准直器宽度0.65mm,层厚5mm）,将含有2ml静脉血的肝素抗凝管4只/组水平并排放置于CT扫描床,对B、C和D管分别行1次,2次和3次扫描。以剂量长度乘积（Dose Length Product,DLP）代表CT检查的辐射剂量。CT扫描后5-10分钟内提取外周血淋巴细胞,行洗涤、固定、破膜、封闭、标抗等一系列操作,配成1ml的悬液,并取20ml细胞悬液均匀涂于防滑脱载玻片,晾干后行染色和DAPI封片处理。荧光显微镜下观察并计数γH2AX焦点,由两位病理科医生使用双盲法计数,选择细胞均匀,背景清晰的区域,每个标本计数50个细胞,两位医生计数的一致性使用kappa检验。剩余悬液以400ml/管分于2个离心管内,管1作为背景管,管2加入二抗并孵育,40分钟后洗涤,并配成400ml/管的悬液上机,设定流式细胞仪自动计数10000个细胞,计数γH2AX阳性细胞的个数,并以每万个细胞内阳性细胞百分比表示,最终γH2AX阳性细胞比例为Hist（试验管%-背景管%）。结果两位病理科医生对免疫荧光γH2AX计数结果的一致性Kappa值为0.52,B-D管CT辐射剂量DLP分别为（226.83±13.22）mGy·cm、（448.5±22.18）mGy·cm和（670.17±31.92）mGy·cm,A-D管免疫荧光法所测定的平均γH2AX焦点数分别为（0.059±0.029）、（0.73±0.351）、（1.211±0.509）和（1.750±0.549）个/细胞,流式细胞法所测得平均γH2AX量分别为（1.009±1.961）%、（3.313±3.778）%、（5.78±5.046）%和（11.294±6.793）%。两者所测定的代表DNA损伤的γH2AX量均与辐射次数成正相关,并且免疫荧光法与流式细胞法定量测得的γH2AX与代表辐射剂量的DLP的相关性r分别为0.63和0.74。结论1.免疫荧光和流式细胞法均可用于CT辐射生物学效应的定量测定,两者所测定的γH2AX量与辐射次数均成正相关。2.免疫荧光法可以更直观显示γH2AX焦点数量和质量,但步骤较为繁琐,容易受到人为因素干扰,两名观察者的一致性不够高（kappa=0.52）。3.流式细胞仪法定量测得的γH2AX与辐射剂量（DLP）的相关性高于免疫荧光法。第二部分碘对比剂血液内环境对CT辐射生物学效应的影响（体外实验）目的评估离体静脉血内碘对比剂的存在对CT辐射致DNA双链损伤的生物学效应影响。材料与方法采集21例志愿者的外周静脉血,12ml/人,平均分6管于肝素抗凝管中（2ml/管）,依次标为1-6号管,1号管为对照管,加入0.1ml生理盐水,2号管加入0.1ml生理盐水,3号管加入0.05ml碘对比剂和0.05ml生理盐水,4号管加入0.1ml碘对比剂,5号管加入0.1ml高浓度对比剂,6号管加入0.05ml对比剂和0.05ml生理盐水。2-5号管并排捆绑成2×2立方体结构,平放于CT扫描床。使用西门子第二代双源CT（Definition Flash,Siemens,德国）,设定每次扫描参数一致（电压120kv,电流210mAs,关闭4D CareDose,扫描FOV 250mm×250mm,长度150 mm,机架旋转速度300ms/rot,准直器宽度0.65mm,层厚5mm）,对2号、3号和4号管分别行2次扫描。以剂量长度乘积（Dose Length Product,DLP）代表CT检查的辐射剂量。CT扫描后5-10分钟使用Ficoll分层液法提取外周血淋巴细胞,行洗涤、固定、破膜、封闭、标抗等一系列操作,最后配成800ml的悬液,并以400ml/管分于2个离心管内,管A作为背景管,管B加入二抗并孵育,40分钟后两管洗涤,并配成400ml/管的悬液后上机测定,设定流式细胞仪自动计数10000个细胞,计数γH2AX阳性细胞的个数,并以每万个细胞内阳性细胞百分比表示,最终γH2AX阳性细胞比例为Hist（试验管%-背景管%）。结果2-5号管平均辐射剂量均为（452.3±26.5）mGy·cm,1-6号管γH2AX定量评估百分比分别为（0.45±0.12）%、（4.40±2.99）%、（6.38±5.77）%、（7.28±7.37）%、（4.36±4.86）%和（4.89±5.65）%,其中3号管γH2AX百分比高于2号管,随着碘对比剂浓度升高,4号管γH2AX量较3号管升高,但碘对比剂浓度增加到0.015ml/管时,γH2AX数量反而下降。6号管为碘对比剂单独作用管,但其γH2AX量明显高于对照管。结论1.碘对比剂的存在对CT辐射的生物学效应有“增强作用”,并且与碘对比剂浓度有关,但当超过一定浓度时（本试验为24.42mgI/ml）,此作用降低。2.碘对比剂可单独导致淋巴细胞内γH2AX升高,即碘对比剂可引起淋巴细胞DNA的单独损伤。第三部分碘对比剂血液内环境对CT辐射生物学效应的影响（体内实验）目的在体评估碘对比剂血液内环境对CT检查致淋巴细胞DNA损伤辐射生物学效应的影响以及碘对比剂自身对淋巴细胞DNA损伤的生物学效应。材料与方法60例临床怀疑泌尿道系统疾病而准备行CTU检查的患者随机分为对照组和实验组。两组患者扫描均分为2次进行CTU扫描:对照组使用常规法CTU检查（包括平扫、皮质期、髓质期和排泄期四期）,第1次仅行泌尿系CT平扫,扫描前和扫描后10分钟内各抽取肘静脉血液2ml,分别标为A、B管,三天后补充常规法CTU增强扫描。实验组使用分次注射法CTU检查,第1次先行分次注射增强扫描（皮髓质-排泄期）（具体注射方案为:先以3 ml/s速率注入50 ml碘对比剂,注射结束后患者静躺于检查床等待,850 s后以同样速率再次注射40 ml对比剂,再30 s后启动实质-排泄期扫描）分别于扫描前、碘对比剂注射10分钟后和扫描后10分钟内抽取肘静脉血,并分别标记为A1、C和B1。分离外周静脉血淋巴细胞、经过4%多聚甲醛固定,加抗体孵育,最后使用流式细胞仪定量测定各管内γH2AX含量。对照分析A-B和A1-B1管内γH2AX含量变化,评估碘对比剂血液内环境对CT辐射致人体外周血淋巴细胞DNA的影响;并且对照分析C管相对与A1管内γH2AX量的变化,评估碘对比剂对淋巴细胞DNA的单独生物学效应。结果对照组和实验组CT扫前γH2AX含量分别为（19.204±5.942）%和（20.217±7.498）%,差异无统计学意义（t=0.11,P=0.94）;扫描后分别为（31.219±7.780）%和（39.260±8.003）%,差异有统计学意义（t=-3.09,P=0.00）;两组扫描前后差值分别为（12.015±2.119）%和（19.043±3.452）%,差别有统计学意义（t=-2.58,P=0.00）,实验组较对照组的γH2AX数量增加了58.49%;对照组的碘对比剂注射前后（10分钟内采集血样）外周静脉血内γH2AX含量为（20.217±7.498）%和（26.100±10.743）%,差异有统计学意义（t=-2.12,P=0.00）,注射对比剂后γH2AX增加了29.08%。结论1.碘对比剂血液内环境可增加CT检查所致人体外周静脉血淋巴细胞内DNA损伤数量,增加幅度约为58.49%;2.碘对比剂本身可引起外周静脉血内淋巴细胞DNA的单独损伤,幅度约为29.08%。第四部分碘对比剂和CT辐射所致人体外周静脉血淋巴细胞DNA损伤后自体修复情况研究目的评估碘对比剂血液内环境和单独CT检查的X射线辐射所致人体外周血淋巴细胞DNA损伤后自体的修复程度,以及两种方法致DNA损伤后的修复-时间差异。材料与方法选择健康志愿者为研究对象,共10例,平均年龄42岁,抽取10名志愿者抽取外周静脉血各16ml,平均分为2ml/管共8管,存入肝素抗凝管中,将8管血样分为A、B两组,每组4管,A1-A4组为碘对比剂组,每管加入50ml碘对比剂（350mgI/ml）,用100ml移液枪直接加入并混匀,操作过程保持无菌。B1-B4管为CT扫描组,每组扫描一次。使用西门子第二代双源CT（Definition Flash,Siemens,德国）,设定每次扫描参数一致（电压120kv,电流210mAs,关闭4D CareDose,扫描FOV 250mm×250mm,长度150 mm,机架旋转速度300ms/rot,准直器宽度0.65mm,层厚5mm）,将含有2ml静脉血的肝素抗凝管4只/组水平并排放置于CT扫描床,对B1-B4管进行1次扫描。以剂量长度乘积（Dose Length Product,DLP）代表CT检查的辐射剂量。碘对比剂注射完毕或扫描结束后将血样存入37℃无菌保温箱,以保持活性,分别于注入碘对比剂或CT扫描后的30min、2h、4h和8h后开始处理血液。使用Ficoll分层液法^[9]提取外周血淋巴细胞,行洗涤、固定、破膜、封闭、标抗等一系列操作,最后配成800ml的悬液,并以400ml/管分于2个离心管内,管A作为背景管,管B加入二抗并孵育,40分钟后两管洗涤,配成400ml/管的悬液后上机测定,设定流式细胞仪自动计数10000个细胞,计数γH2AX阳性细胞的个数,并以每万个细胞内阳性细胞百分比表示,最终γH2AX阳性细胞比例为Hist（试验管%-背景管%）。结果A1-A4管碘对比剂注入顺利（50ml/2ml）,B1-B4号管平均辐射剂量均为（234.25±14.11）mGy·cm。在30min、2h、4h和8h后A1-A4号管γH2AX定量评估百分比分别为（38.07±21.29）%、（43.50±25.52）%、（33.60±10.46）%、（28.31±8.69）%;B1-B4管分别为（34.29±19.92）%、（49.27±16.72）%,（14.21±10.70）%、（7.82±3.49）%。两组均在2h时γH2AX达到最大量,但4小时后B组的γH2AX量明显低于A组。结论碘对比剂和CT辐射导致的外周静脉血淋巴细胞DNA损伤后,机体有自我修复能力,2小时修复达到高峰,但4小时后,碘对比剂所致的损伤γH2AX数量明显高于CT辐射。更多还原

【Abstract】 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.更多还原