人外周血网织红细胞微核的流式细胞仪自动化检测

背景与目的:建立一种基于单激光流式细胞仪的人外周血网织红细胞微核率自动化检测方法.材料与方法:在本室已建立的大鼠骨髓嗜多染红细胞微核流式细胞仪自动化检测体系基础上,使用磁式细胞分选技术,富集并分离人外周血中的网织红细胞,采用吖啶橙(acridine orange,AO)荧光染色,用流式仪检测人外周血中的网织红细胞微核率.结果:磁式细胞分选技术分离到的CD71阳性红细胞占网织红细胞总量的20%至90%,产量在不同个体之间存在显著差异,但同一个体其分离效果几乎没有差异;网织红细胞经过富集后的人外周血样本被分成明显的五个细胞群--网织红细胞、含微核的网织红细胞、成熟红细胞、含微核的成熟红细胞以及有核细胞;7名健康人外周血网织红细胞微核率平均值为2.36‰,10名接受累积放疗剂量0、4、10、20、30、40及50Gy后的鼻咽癌患者外周血网织红细胞微核率平均值分别为2.97‰、29.37‰、57.90‰、69.05‰、62.58‰、51.19‰及50.07‰.结论:成功建立了基于单激光流式细胞仪的人外周血网织红细胞微核率自动化检测体系.     

肿瘤患者放、化疗后外周血淋巴细胞损伤和免疫功能改变的研究

目的观察肿瘤患者放、化疗后外周血淋巴细胞损伤和免疫功能改变,并分析两者之间的关系.方法30例肿瘤患者分成放射治疗组(放疗组)和化学药物治疗组(化疗组),分别在放疗前后,化疗前后测定外周血双核淋巴细胞微核率(‰)、T淋巴细胞转化率(%)及血清免疫球蛋白(IgG,IgA,IgM),并进行对比分析.双核淋巴细胞微核率(MNR)用细胞松驰素B(CB)法测定,T淋巴细胞转化率(TLTR)及免疫球蛋白(IgG,IgA,IgM),均用常规方法测定.结果放疗组和化疗组在放、化疗后外周血双核淋巴细胞微核率显著性升高,而T淋巴细胞转化率和血清免疫球蛋白(IgG,IgA,IgM)均显著性降低.结论放、化疗后,两组患者的外周血双核淋巴细胞微核率(‰)与T淋巴细胞转化率(%)呈负相关关系.     

放射治疗前后鼻咽癌患者外周血淋巴细胞微核率改变的研究

目的探讨放射治疗对鼻咽癌(na-sopharyngeal carcinoma,NPC)患者正常组织的损伤效应,为判断正常组织放射反应提供敏感的生物学指标。方法在NPC患者接受放射治疗前后分别采用常规培养法和胞质分裂阻滞法进行外周血淋巴细胞微核率检测,选择健康人作为对照组。结果NPC患者放疗前常规培养法和胞质分裂阻滞法的淋巴细胞微核率分别是3·13×10-3、15·20×10-3,显著高于对照组1·20×10-3、9·00×10-3,P<0·01;接受放射治疗后1例患者除外,其余患者微核率增高,与放疗前相比差异有统计学意义,P<0·05。结论NPC患者本身染色体稳定性较健康人差,肿瘤部位接受射线治疗的同时,机体正常组织也受到了损伤。     

鼻咽癌放疗患者细胞微核分析

微核试验作为细胞遗传学损伤的生物标志之一, 是公认的检测染色体异常的简便方法, 在医学领域中正得到越来越广泛的应用。X2射线放疗是鼻咽癌患者最主要的治疗方法。本文采用微核试验方法观察鼻咽癌放疗患者口腔颊粘膜脱落细胞及人外周血淋巴细胞微核率的变化, 以探讨微核作为正常细胞损害和受照剂量生物标志物的可能性。检测对象为10 例接受X2射线放疗的鼻咽癌患者, 均来自第三军医大学附属西南医院放疗中心, 患者平均年龄37 岁, 男女各5 例, 照射总剂量平均为68 GY。本次实验采用病人为自身对照。分别于放疗前, 照射2、5、14、24、34 次后(每次限射剂量为2 GY) , 各采血一次用于实验。口腔颊粘膜脱落细胞微核试验: 取口腔颊粘膜脱落细胞直接涂片, 固定后用吖啶橙染色, 荧光显微镜下观察1 000 个脱落细胞中的微核细胞数。人外周血淋巴细胞微核试验采用胞质分裂阻滞法微核试验, 取0. 5 m l 静脉血于4. 5 m l RPM I 1640 培养基中37℃培养44 h 加入6 mg·L - 1的松胞素B, 继续培养至72 h 收获细胞, 常规制片。微核判断标准: 在淋巴细胞胞质中, 与主核分开, 呈圆形或椭圆形, 边缘光滑, 折光性与主核一致或稍浅, 大小为主核的1ö3～ 1ö的小核。每例记数1 000 个细胞, 结果用千分率表示。结果: 口腔颊粘膜脱落细胞微核率放疗前后未观察到明显变化。外周血淋巴细胞第一次放疗后微核率即有显著升高, 以后随剂量的增加几乎成线性升高。结论: 一定剂量的X2射线照射后, 对鼻咽癌患者口腔颊粘膜无明显损伤作用, 而外周血淋巴细胞微核检出率则非常敏感, 并呈正相关的剂量-反应关系, 有可能作为受照剂量的一个备选生物标志物。     

71. 鼻咽癌放疗患者细胞微核分析

微核试验作为细胞遗传学损伤的生物标志之一,是公认的检测染色体异常的简便方法,在医学领域中正得到越来越广泛的应用。X-射线放疗是鼻咽癌患者最主要的治疗方法。本文采用微核试验方法观察鼻咽癌放疗患者口腔颊粘膜脱落细胞及人外周血淋巴细胞微核率的变化,以探讨微核作为正常细胞损害和受照剂量生物标志物的可能性。检测对象为10例接受X-射线放疗的鼻咽癌患者,均来自第三军医大学附属西南医院放疗中心,患者平均年龄37岁,男女各5例,照射总剂量平均为68 GY。本次实验采用病人为自身对照。分别于放疗前,照射2、5、14、24、34次后（每次限射剂量为2 GY）,各采血一次用于实验。口腔颊粘膜脱落细胞微核试验：取口腔颊粘膜脱落细胞直接涂片,固定后用吖啶橙染色,荧光显微镜下观察1 000个脱落细胞中的微核细胞数。人外周血淋巴细胞微核试验采用胞质分裂阻滞法微核试验,取0.5 ml静脉血于4.5 ml RPMI 1640培养基中37℃培养44 h加入6 mg*L-1的松胞素B,继续培养至72 h收获细胞,常规制片。微核判断标准：在淋巴细胞胞质中,与主核分开,呈圆形或椭圆形,边缘光滑,折光性与主核一致或稍浅,大小为主核的1／3～1／的小核。每例记数1 000个细胞,结果用千分率表示。结果：口腔颊粘膜脱落细胞微核率放疗前后未观察到明显变化。外周血淋巴细胞第一次放疗后微核率即有显著升高,以后随剂量的增加几乎成线性升高。结论：一定剂量的X-射线照射后,对鼻咽癌患者口腔颊粘膜无明显损伤作用,而外周血淋巴细胞微核检出率则非常敏感,并呈正相关的剂量-反应关系,有可能作为受照剂量的一个备选生物标志物。     

正常人外周血淋巴细胞体外辐射敏感性的研究

本文利用PHA和白细胞介素2结合克隆的形成测定了人外周血新鲜淋巴细胞体外辐射敏感性。外周血淋巴细胞来自健康成年人,在体外照射x射线0～5Gy,并获得了剂量活存曲线,D-(10)值(杀伤90％细胞时所需剂量)约为3.5Gy。 另一种方法是用细胞松弛素B(CB)阻断细胞浆分裂,从体外受照淋巴细胞形成双核微核率的结果,得出双核微核形成率和受照剂量(0～3Gy)之间呈良好线性关系。     

外周血淋巴细胞松胞素阻滞微核法预测鼻咽癌放射敏感性的临床和实验研究

目的探讨用外周血松胞素阻滞微核法来预测鼻咽癌放射敏感性的可行性。方法前瞻性研究42例鼻咽癌患者放疗前照射0,0.5,1,2,4Gy的外周血,放疗20Gy、60Gy时的外周血行松胞素阻滞微核法检测微核率、微核细胞率,统计分析正常组织敏感组与不敏感组间、鼻咽肿瘤组织局控组与残留组间微核率、微核细胞率、50Gy鼻咽肿瘤消退率等指标有无差异,并分析这些指标间的相互关系。结果在体外照射1Gy及以上剂量、20Gy、60Gy时外周血微核率、微核细胞率在放射敏感组明显比不敏感组增加,在体外照射2Gy及以上剂量、20Gy、60Gy时外周血微核率、微核细胞率在肿瘤局控组比未局控组明显增加。结论外周血淋巴细胞松胞素阻滞微核法有可能作为预测鼻咽癌皮肤、黏膜急性反应敏感性的指标。外周血淋巴细胞微核率、微核细胞率有可能作为预测鼻咽癌根治性放疗近期疗效的指标。     

外周血网织红细胞和T淋巴细胞亚群用于辐射损伤快速剂量估算的可行性研究

目的：通过观察受不同剂量照射小鼠的外周血网织红细胞的百分率变化以及辅助T淋巴细胞（helperT cells,Th）与抑制T淋巴细胞（suppressor T cells,Ts）的比值（rTh/Ts））和照射剂量的关系,探索其用于辐射损伤生物剂量估算的可行性。方法：C57BL/6小鼠,7～8周龄,60Coγ线全身照射0～7Gy,分别收集辐照前和辐射后不同时间点的静脉血,流式细胞术（FCM）检测外周血中网织红细胞百分率和rTh/Ts）情况,分析时间-效应关系并拟合剂量-效应曲线。结果：照射剂量在0～7 Gy范围内,小鼠的外周血网织红细胞百分率均随时间而降低,并于照射后3 d降到最低,且剂量越大恢复越晚;照射后72 h内小鼠的外周血rTh/Ts）均随剂量增加而递增。照射剂量在1～3 Gy范围内,在照射后24、48和72h,小鼠的外周血网织红细胞百分率均随剂量增加而递减;照射剂量在1～7Gy范围内,在照射后6、24和72 h,小鼠的外周血rTh/Ts）均随剂量增加而递增。网织红细胞百分率和_（Th/Ts）的剂量-效应关系均满足直线模型。结论：FCM检测网织红细胞百分率和rTh/Ts）可成为早期快速、高通量的辐射损伤生物剂量计。     

恶性肿瘤患者放疗后胞质分裂阻断的淋巴细胞微核率的变化

我们采用胞质分裂附断微核法对１８例肿瘤放疗患者的淋巴细胞微核进行了检测分析。其中食管癌８例、肺癌４例、乳腺癌３例，其它肿瘤３例。微核检测分三次，即放疗前累积剂量达到３０Ｇｙ和６０Ｇｙ时。结果显示患者的平均微核率分别为１５．６±６．６‰、６５．１±１９．０‰、１２２．６±３５．８‰。所有患者血淋巴细胞微核率与照射剂量之间呈良好的线性关系。接受同等剂量照射的三组患者其组间微核率无明显差异。     

大鼠骨髓嗜多染红细胞微核的流式细胞仪自动化检测

背景与目的:建立一种基于单激光流式细胞仪的大鼠骨髓嗜多染红细胞微核率自动化检测方法.材料与方法:以环磷酰胺诱导雄性Wistar大鼠微核形成,采用吖啶橙(acridine orange,AO)荧光染色,分别用荧光显微镜及单激光流式细胞仪检测大鼠骨髓嗜多染红细胞微核率及正染红细胞微核率.结果:以前置角散射光(FSC)和DNA荧光(FLl)作图,大鼠骨髓细胞被分成明显的五个细胞群--有核细胞、嗜多染红细胞、正染红细胞、含微核的嗜多染红细胞及含微核的正染红细胞;经环磷酰胺处理的大鼠骨髓嗜多染红细胞微核率及正染红细胞微核率均呈现良好的剂量-反应关系及时效关系;人工显微镜计数结果与流式细胞仪检测结果具有良好的相关性(R=0.949,P＜0.01),二者的曲线拟合方程为Y=1.0967 1.0639X(R=0.949,P＜0.01).结论:本研究建立了AO染色结合单激光流式细胞仪的大鼠骨髓嗜多染红细胞微核率自动化检测方法.     

Micronucleated erythrocytes as an index of cytogenetic damage in humans: demographic and dietary factors associated with micronucleated erythrocytes in splenectomized subjects

Erythrocytes containing micronuclei serve as an indicator of genotoxic exposure in splenectomized individuals. Micronucleated erythrocytes, derived from cytogenetically damaged RBC precursors, are not selectively removed from peripheral blood in individuals who lack splenic function. The relationship between micronucleated cell frequencies and demographic, environmental, and dietary factors was examined in 44 subjects with previous splenectomy due to trauma. Their micronucleated cell counts fit a log-normal distribution, with geometric means of 3.3 micronucleus-containing cells/1000 reticulocytes and 2.7/1000 normochromatic erythrocytes. A multiple regression analysis showed that drinking five cups of coffee or tea/day (relative to none) was associated with an approximately 2-fold higher frequency of micronucleated cells. Weaker statistical associations were also noted with micronucleus frequency and the consumption of calcium supplements (associated with a higher frequency) and vitamins A, C, or E (lower frequency). An apparent trend of higher micronucleus counts with age was attenuated when other factors were considered in the regression. Cigarette smoking and decaffeinated coffee consumption were among the factors not associated with elevated micronucleated cell frequencies. Because the occurrence of micronuclei in reticulocytes reflects cytotoxic exposures within the past 3-8 days, it may be possible to test directly the relationship of these factors to micronucleus formation through intervention studies.     

X线放射治疗对鼻咽癌患者外周血淋巴细胞DNA损伤的研究

背景与目的: 评价X线放射治疗对鼻咽癌患者外周血淋巴细胞DNA的损伤。 材料与方法: 采用碱性单细胞凝胶电泳技术,检测19例鼻咽癌X线放射治疗患者在累积照射剂量为0、2、4、8、10、16 Gy时,外周血淋巴细胞的拖尾率,尾长及Olive尾距。 结果: 鼻咽癌患者接受X线放射治疗后,淋巴细胞拖尾率、尾长在累积照射剂量为2 Gy时即出现明显上升(P<0.01 和 P<0.05),Olive尾矩在累积照射剂量为8 Gy时出现明显上升(P<0.01)。在0～16 Gy照射剂量范围内随着剂量的升高外周血淋巴细胞的拖尾率,尾长及Olive尾距随之增加,且呈剂量-反应关系。 结论: 在本实验条件下,X线放射治疗在低剂量时即对鼻咽癌患者外周血淋巴细胞DNA有损伤作用,其照射剂量与外周血淋巴细胞的拖尾率,尾长及Olive尾距呈剂量-反应关系。     

苦参素对小鼠的急性毒性和外周血红细胞微核和精子畸变试验

背景与目的： 研究苦参素的急性毒性及遗传毒性。材料与方法： 采用LD50试验,外周血微核试验与精子畸变试验,按体重随机分为五组,以腹腔注射（i.p）方式给药。结果： 小鼠苦参素的LD50为(505±31)mg/kg。外周血24 h PCE、48 h PCE微核率显示：1/2 LD50、1/4 LD50、1/8 LD50组与阴性对照组相比均有统计学意义,且有一定的剂量反应关系。精子畸变试验中1/2 LD50组的畸变率与阴性对照组相比有统计学意义,1/4 LD50、1/8 LD50组与阴性对照组相比无统计学意义。结论： 苦参素基本属低毒类药物,在一定程度上有遗传毒性作用。     

双灵固本散对肿瘤放化疗后外周血淋巴细胞及免疫功能损伤的防护作用

目的　观察双灵固本散对肿瘤患者放疗、化疗后外周血淋巴细胞及其免疫功能损伤的防护作用。方法　 60例肿瘤患者分成 4个组 :放疗对照组 ,放疗辅加双灵固本散治疗组 (双灵放疗组 ) ,化疗对照组 ,化疗辅加双灵固本散治疗组 (双灵化疗组 )。采用CB法测定外周血双核淋巴细胞微核率 (MNR)、T淋巴细胞转化率 (TLTR )及IgG、IgA、IgM水平的变化。 结果　放疗对照组和化疗对照组MNR显著性升高 ,而TLTR和IgG、IgA、IgM水平均显著降低。双灵放疗组和双灵化疗组MNR、TLTR和IgG、IgA、IgM水平均无显著性变化。放疗对照组、化疗对照组患者放疗、化疗后外周血MNR与TLTR呈负相关关系。 结论　双灵固本散明显减轻肿瘤患者放疗、化疗后T淋巴细胞核DNA及其免疫功能损伤     

Micronuclei in exfoliated human cells as a tool for studies in cancer risk and cancer intervention

The use of the micronucleus test on exfoliated cells as an approach to identify genotoxic damage in human tissues which are targets for organ-specific carcinogens and from which carcinomas will develop, is described. Chromosomal damage by carcinogens to dividing basal cells of the epithelium results in the production of micronuclei in the daughter cells which migrate up through the epithelium and are exfoliated. Exfoliated cells can be readily obtained from several tissues, including the oral buccal mucosa (scrapings of oral cells), bronchi (sputum), urinary bladder and ureter (centrifugation of urine), cervix (smears) and esophagus (imprints from biopsies). The micronucleus test on exfoliated cells has been successfully used to: (1) recognize population groups at an elevated risk for cancer of the oral cavity or urinary bladder; (2) estimate synergistic or additive effects of carcinogen exposure (cigarette smokers plus drinkers of alcoholic beverages); (3) pinpoint the site within an organ from which most carcinomas will develop (oral cancers among 'inverted' smokers in the Philippines). The possibility that this assay may also serve as a rapid monitor for chemopreventive agents is suggested by a preliminary trial on the effect of vitamin A/beta--carotene dietary supplementation among 33 betel quid chewers in the Philippines. These individuals received sealed capsules of retinol (100,000 IU/week) and beta-carotene (300,000 IU/week) for a 3-month period. At the end of this time, the frequencies of micronucleated buccal mucosa cells were reduced from an average of 4.2% to 1.4%. No changes were observed in micronucleus frequencies among 11 betel quid chewers not receiving vitamin pills. Non- chewers of betel quid in this population had a micronucleus frequency of 0.5%.     

人外周血淋巴细胞微核分析用于估算离体模拟局部照射剂量的研究

目的：探讨利用人外周血淋巴细胞微核分析估算局部照射剂量的可行性。方法：收集2例人外周血样本,每例血样分为两部分,一部分不照射,另一部分再分成两组分别于1和5 Gy的⁶⁰Co γ射线下离体照射,剂量率为1 Gy/min。将来自同一样本的照射血与未照射血按1∶3或3∶1比例混合以模拟局部照射,共分析8组混合血样中双核淋巴细胞微核（MN）率,利用国际原子能机构（IAEA）推荐的Dolphin's模型推算混合血中受照血的微核率,并采用卫生行业标准（WS/T 178—1999）推荐的剂量效应曲线估算局部照射剂量。结果：各组混合血样MN分布均不符合泊松分布。1 Gy照射组估算的局部剂量与实际照射剂量偏差较大,5 Gy照射组估算的局部剂量与实际照射剂量较接近。结论：离体情况下,MN能较好的用于估算局部照射剂量,且MN可能更适用于较高剂量的估算。     

Development of gene expression signatures for practical radiation biodosimetry

PURPOSE: In a large-scale radiologic emergency, estimates of exposure doses and radiation injury would be required for individuals without physical dosimeters. Current methods are inadequate for the task, so we are developing gene expression profiles for radiation biodosimetry. This approach could provide both an estimate of physical radiation dose and an indication of the extent of individual injury or future risk. METHODS AND MATERIALS: We used whole genome microarray expression profiling as a discovery platform to identify genes with the potential to predict radiation dose across an exposure range relevant for medical decision making in a radiologic emergency. Human peripheral blood from 10 healthy donors was irradiated ex vivo, and global gene expression was measured both 6 and 24 h after exposure. RESULTS: A 74-gene signature was identified that distinguishes between four radiation doses (0.5, 2, 5, and 8 Gy) and controls. More than one third of these genes are regulated by TP53. A nearest centroid classifier using these same 74 genes correctly predicted 98% of samples taken either 6 h or 24 h after treatment as unexposed, exposed to 0.5, 2, or > or =5 Gy. Expression patterns of five genes (CDKN1A, FDXR, SESN1, BBC3, and PHPT1) from this signature were also confirmed by real-time polymerase chain reaction. CONCLUSION: The ability of a single gene set to predict radiation dose throughout a window of time without need for individual pre-exposure controls represents an important advance in the development of gene expression for biodosimetry.     

Quantitating the synergistic effect of smoking and alcohol consumption with the micronucleus test on human buccal mucosa cells

The micronucleus test was applied to exfoliated cells of the buccal mucosa of four population groups: (A) non-smokers and non-drinkers of alcoholic beverages, (B) non-smokers but alcohol drinkers, (C) smokers but non-drinkers, and (D) smokers and drinkers. An elevated frequency of micronucleated buccal mucosa cells was observed only in group D (smokers and alcohol drinkers). When group D was subdivided according to the number of cigarettes smoked, the frequency of micronucleated buccal cells and the average number of micronuclei per cell appeared to depend upon cigarette consumption. An approximately eight-fold increase of micronucleated mucosa cells was seen among alcohol drinkers who smoked three or more packs of cigarettes per day, and an approximately 4.2-fold elevation was observed when one to two packs were consumed. Neither smoking alone of up to and over 60 cigarettes per day nor ethanol drinking alone of up to 1.21 per day led to a detectable elevation of micronucleated buccal mucosa cells. Whether the strong synergistic effect between smoking and alcohol consumption, as seen by the frequency of micronucleated buccal mucosa cells, is related to their synergistic effect in the induction of oral cancers is an intriguing but open question.     

Analysis of the results of the micronucleus test in patients presenting upper digestive tract cancers and in non-cancerous subjects

A micronucleus test was performed on 75 subjects of whom 38 presented with cancer of the upper digestive tract and 37 were free of disease; the absence of cancerous or pre-cancerous lesions in this latter group was confirmed by endoscopy and vital staining. The daily levels of alcohol and tobacco consumption of the 75 subjects were determined by precise questioning: 78% of the non-cancerous subjects smoked less than 10 g of tobacco per day whereas 79% of the cancer patients smoked 10 g or more daily. The alcohol intake of 78% of the non-cancerous subjects and 63% of the cancer patients was less than 101 ml per day. Only 10% of the cancer patients had combined daily intake levels corresponding to the threshold of sensitivity of the micronucleus test as defined by previous studies. The mean frequency of micronucleated buccal cells was 0.26% in the cancer patients and 0.13% in the non-cancerous subjects. All non-cancerous patients presented a negative test. Only 5% of the cancer patients presented a micronucleated cell frequency above 1% and could thus be considered as positive. It thus appears that the micronucleus test was not significantly positive in our population of 38 cancer patients.