辐射诱发淋巴细胞凋亡生成与抑制作用研究

研究了辐射诱发的人外周血淋巴细胞凋亡生成，以及水溶性维生素Ｅ类似物－Ｔｒｏｌｏｘ对辐射诱导人外周血淋巴细胞凋亡的抑制作用。照后３０分钟内Ｔｒｏｌｏｘ能有效地阻抑ＤＮＡ片段形成，而在照前或受照中加入Ｔｒｏｌｏｘ均不能抑制ＤＮＡ片段形成，揭示Ｔｒｏｌｏｘ并不是通过清除照射过程中产生的自由基而起作用。照后３０分钟内加Ｔｒｏｌｏｘ，２小时后撤去，同样能抑制ＤＮＡ片段形成，表明Ｔｒｏｌｏｘ能不可逆地阻抑细胞凋亡早期的＂关键＂事件。     

5例放射事故病人照后5.5年外周血淋巴细胞 HPRT 基因突变频率和突变谱的测定

通过克隆外周血淋巴细胞并用６巯基鸟嘌呤筛选次黄嘌呤磷酸核糖转移酶基因（ｈｙｐｏｘａｎｔｈｉｎｅｐｈｏｓｐｈｏｒｉｂｏｓｙｌｔｒａｎｓｆｅｒａｓｅ，ＨＰＲＴ）缺陷的突变细胞，测定了５例急性放射病人在受２．０～５．２Ｇｙ６０Ｃｏγ射线意外照射后５．５年外周血淋巴细胞ＨＰＲＴ基因突变频率。观察到细胞克隆效率、突变细胞克隆数、ＨＰＲＴ突变频率与受照剂量有一定的依赖性。用多引物聚合酶链反应（ＰＣＲ）扩增突变细胞ＨＰＲＴ基因全部９个外显子伴以琼脂糖凝胶电泳以分析基因突变谱。结果表明，高剂量受照病人的外显子缺失总数较低剂量者高。与照后３．５年和４．５年的突变谱比较，外显子缺失突变比例逐年下降，而点突变比例逐年升高。可能反映病人机体的恢复和受损ＤＮＡ的修复。     

X射线对小鼠胸腺细胞凋亡的影响

采用荧光分光光度法对裂解ＤＮＡ进行定量，并用琼脂糖凝胶电泳法定性研究Ｘ射线全身照射后小鼠胸腺细胞凋亡。结果表明，４ＧｙＸ射线照后２小时胸腺细胞凋亡开始增加，于照后１４小时达峰值，尔后呈下降趋势。ＤＮＡ裂解率在照后２４小时降至１４小时的５０％，这可能是内环境中巨噬细胞吞噬凋亡小体所致。在照射后１４小时研究其剂量－效应关系发现，高剂量照射使ＤＮＡ裂解明显增多，形成１８０ｂｐ（ｂａｓｅｐａｉｒｓ）左右或其整倍数的ＤＮＡ断片，电泳呈现＂梯形图谱＂；而低剂量辐射可使ＤＮＡ裂解率降低。剂量－效应曲线呈Ｊ型。     

^60Co辐射事故5名受照者外周血T细胞抗原受体基因的…

在低熔点琼脂糖胶内制备人淋巴细胞基因组ＤＮＡ，两种限制性核苷酸内切酶对低熔点胶块内的样本ＤＮＡ进行完全消化，以人Ｔ淋巴细胞抗原受体（ＴＣＲ）的α和β链ｃＤＮＡ为探针，对５名４．５年前全身受＾６０Ｃｏ大剂量受照者外周血淋巴细胞基因组ＤＮＡ进行印迹杂交分析。结果发现经ＨｉｎｄⅢ内切酶消化的样本ＤＮＡ，正常对照与受照者比较，两种ｃＤＮＡ探针的杂交结果无明显区别，经ＥｃｏＲＩ内切酶消化的ＤＮＡ样本，４名受     

五例60Co源辐射事故病人照后3.5～5.5年外周血淋巴细胞HPRT基因突变谱分析

目的对５例受２．０～５．２Ｇｙγ射线照射的事故病人照后３．５～５．５年外周血淋巴细胞ＨＰＲＴ基因突变谱进行监测。方法用多引物聚合酶链式反应扩增突变细胞ＨＰＲＴ基因的全部外显子伴以琼脂糖凝胶电泳以分析基因突变谱。结果外显子缺失是电离辐射所致ＨＰＲＴ基因突变的主要特征。随着时间推移，外显子缺失总数由照后３．５年的１８／２４（７５％）下降至４．５年的３１／４６（６７．４）和５．５年的１２／３０（４０％）；而点突变比例则相应地上升。这可能反映了ＤＮＡ损伤的修复和病人机体的恢复。结论ＨＰＲＴ基因突变在体内长期存在，但基因突变谱逐渐发生值得注意的变化，随访观察仍应继续     

60Co辐射事故5名受照者外周血T细胞抗原受体基因的印迹杂交分析

在低熔点琼脂糖胶内制备人淋巴细胞基因组ＤＮＡ，两种限制性核苷酸内切酶对低熔点胶块内的样本ＤＮＡ进行完全消化，以人Ｔ淋巴细胞抗原受体（ＴＣＲ）的α和β链ｃＤＮＡ为探针，对５名４．５年前全身受６０Ｃｏ大剂量受照者外周血淋巴细胞基因组ＤＮＡ进行印迹杂交分析。结果发现经ＨｉｎｄⅢ内切酶消化的样本ＤＮＡ，正常对照与受照者比较，两种ｃＤＮＡ探针的杂交结果无明显区别，经ＥｃｏＲⅠ内切酶消化的ＤＮＡ样本，４名受者的αｃＤＮＡ探针杂交带型不同于正常对照。作者对这一结果进行了初步分析。     

ADPRT抑制剂对人肿瘤细胞的放射增敏效应

目的：从细胞的克隆形成能力和细胞ＤＮＡ双链断裂及修复几方面探讨了ＡＤＰ－核糖基转移酶（ＡＤＰＲＴ）的特异性抑制剂３－氨基苯甲酰胺（３－ＡＢ）对人卵巢癌细胞株ＨＯＣ８的放射增敏效应。结果表明，３－ＡＢ能降低受照细胞的克隆形成能力；照射所诱发的初始ＤＮＡ双链断裂水平不受３－ＡＢ的影响，但细胞对双链断裂的修复能力受到抑制，表现为慢速修复水平下降，两方面的结果呈正相关。结论：通过脉冲电场凝胶电泳测定ＤＮＡ双链断裂及其修复水平，可以预测细胞的放射敏感性。     

小鼠受亚致死剂量60Co γ 射线照射后胸腺细胞凋亡的研究

目的探讨小鼠受亚致死剂量６０Ｃｏγ射线照射后胸腺细胞凋亡的变化规律，为研究辐射后免疫功能的重建打下基础。方法采用６０Ｃｏγ射线，２．０，４．０，６．０Ｇｙ单次全身照射，用ＰＩ染色流式细胞仪分析，ＤＮＡ琼脂糖凝胶电泳及细胞激活增殖能力的测定等方法观察胸腺细胞凋亡的规律。结果胸腺细胞照后２小时即出现明显的细胞凋亡，４～８小时达高峰，后渐减少，４．０Ｇｙ，６．０Ｇｙ在３６小时内的凋亡率均比２Ｇｙ高，照后１０天三个剂量组的凋亡率基本接近正常；ＤＮＡ琼脂糖凝胶电泳有大量的小分子片断，呈典型的梯状图谱；４Ｇｙ组用ＰＨＡ、ｒＩＬ－２不同组合刺激受照组细胞，发现胸腺细胞凋亡率有不同程度的下降。结论亚致死剂量６０Ｃｏγ射线照射后小鼠胸腺细胞出现明显的凋亡，４Ｇｙ照射剂量可能是胸腺细胞达凋亡高峰的最适剂量，ＰＨＡ、ｒＩＬ－２联合对辐射诱导的胸腺细胞的凋亡有明显的保护作用。     

急性放射病患者恢复期外周血淋巴细胞表型及功能分析

了解急性放射病患者恢复期间外周血淋巴细胞功能。方法用流式细胞仪分析淋巴细胞表型，３Ｈ－ＴｄＲ掺入法分别分析Ｔ细胞和ＮＫ细胞功能。结果照后４．５年，患者外周血ＣＤ＋４Ｔ细胞仍有不同程度低于正常对照；受照剂量大于２Ｇｙ者ＣＤ＋８Ｔ细胞数明显高于正常，而受照时年龄为５４岁者ＣＤ＋８Ｔ细胞明显低于对照；多数患者外周血ＮＫ细胞数和功能都高于正常对照。结论受照者外周血Ｔ细胞功能恢复与受照剂量大小及年龄因素有关，恢复期ＮＫ细胞数量和活性增高，对于弥补Ｔ细胞功能不足具有积极意义     

碱性单细胞凝胶电泳检测辐射诱发的外周血淋巴细胞DNA损伤

电离辐射往往通过ＤＮＡ损伤而诱发细胞突变和癌变。由Ｓｉｎｇｈ等改进和建立的碱性单细胞凝胶电泳试验(ＳＣＧＥ),是一种检测哺乳动物单细胞ＤＮＡ断裂的新技术[1,2]。作者应用ＳＣＧＥ检测了受γ射线照射后小鼠及从事Ｘ射线探伤工人的外周血淋巴细胞ＤＮＡ单链断裂(ＤＮＡｓｓｂ)的情况。一、材料和方法1实验动物及照射:雄性昆明系小鼠购自原苏州医学院动物部,6～7周龄,体重(20±2)ｇ。用60Ｃｏ外照射治疗机全身照射小鼠。球靶距70ｃｍ,吸收剂量0.5和5.0Ｇｙ,吸收剂量率为90.3ｃＧｙ·ｍｉｎ-1。2人群检测对象:选择从事Ｘ射线探伤的工人1…     

Induction of apoptotic cell death in rat thymus and spleen after a bolus injection of methamphetamine

We examined whether methamphetamine (MAP) induced apoptotic cell death in vivo. Male Wistar rats were injected intraperitoneally with 25 mg MAP/Kg body weight and were sacrificed at 4, 8 and 24 h. As early as 4 h after a single dose of MAP, DNA ladder bands representing DNA fragmentation into multiples of the internucleosomal DNA length of about 180 by were observed by gel electrophoresis in thymic and splenic DNA. DNA from control rats injected with 1 ml physiological saline/Kg body weight showed no ladder band patterns. The proportion of fragmented DNA from the thymus increased in a time-dependent manner up to 8 h and faint ladder band patterns were observed at 24 h, indicating that cell death via apoptosis occurred at an early stage and then apoptotic bodies were scavenged. DNA fragmentation in the thymus and spleen induced with MAP was also confirmed by the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) method in situ. In control thymus samples, stained cells were numerous in the cortex but sparse in the medulla. At the boundary area between the cortex and medulla, stained cells were seen as a layer. In the MAP-treated rats, stained cells were increased and dispersed equally in the cortex and medulla. In control spleen samples, stained cells were numerous in all areas excluding the germinal centers. Cells at the germinal centers were stained intensively in MAP-treated rat spleen. Light microscopical analyses allowed us to identify lymphocytes during the course of apoptotic cell death. Electron microscopic studies showed morphological landmarks for the process of cellular apoptosis in both organs e.g. lymphocytes with chromatin condensed into crescents at the periphery of the nuclei and apoptotic bodies. These results indicate that MAP induced cell death of the thymic and splenic lymphocytes via apoptosis.     

X射线诱导多形性胶质母细胞瘤细胞系的凋亡

辐射诱导肿瘤细胞的凋亡是近年来放射生物学研究的热点，为了进一步探讨辐射诱导肿瘤细胞凋亡的机制，采用光镜、电镜及ＤＮＡ琼脂糖凝胶电泳法观察Ｘ射线诱导多形性胶质母细胞瘤细胞系凋亡的特征。结果表明：①剂量率和分次照射组（１０Ｇｙ）诱导出典型细胞凋亡的特征（如凋亡小体，ＤＮＡ梯状带等）。②剂量诱导肿瘤细胞凋亡，高剂量导致肿瘤细胞坏死。③凋亡出现时间因组织细胞类型的不同而各有差异。④射线诱导肿瘤细胞的凋亡以     

二硫代氨基甲酸吡咯烷诱导人肝癌细胞凋亡的铜离子依赖性机制

目的：测定二硫代氨基甲酸吡咯烷（PDTC）处理对人肝癌细胞Hep3B凋亡的诱导作用及Cu^2 对这种作用的影响。方法：运用细胞增殖力、膜损伤、DNA片断化、细胞周期DNA含量测定分析PDTC对人肝癌细胞株Hep3B细胞的增殖抑制及凋亡诱导作用及Cu^2 在其中发挥的作用。结果：PDTC处理后，细胞增殖能力显著下降。乳酸脱氢酶（LDH）分析显示细胞膜并没有被破坏，DNA凝胶电泳及细胞周期DNA含量测定发现明显的凋亡特征性DNA片断及亚G1峰，显示细胞增殖能力的降低源于细胞凋亡的发生。低浓度Cu^2 显著加强了PDTC的细胞增殖抑制及凋亡诱导作用，而Cu^2 络合剂Bathocuproine disulfonate(BCS)显著抑制PDTC的两种作用。结论：PDTC铜离子依赖性地抑制人肝癌细胞增殖并诱导细胞凋亡。     

茶多酚诱导肝癌细胞凋亡

为进一步研究茶多酚诱导体外培养的肝癌细胞株HepG-II细胞发生凋亡的作用,采用MTT法、形态学观察、琼脂糖凝胶电泳和末端脱氧核苷酸转移标记法观察被茶多酚处理后的ＨepG－II细胞的形态学和生化等指标的变化。MTT法研究结果显示,当茶多酚浓度为250μg/ml时即时诱导HepG-II细胞凋亡,并与浓度呈正相关;当茶多酚浓度＞2000μg/ml时,抑制率增强的幅度明显减慢。透射电镜下观察到核染色质浓集呈块并可见凋亡小体;荧光染色在荧光显微镜下可见部分细胞核或细胞质内出现致密浓染的黄绿色块状和颗粒状荧光等凋亡细胞的形态学改变。琼脂糖凝胶电泳呈典型的DNA梯形图像。末端脱氧核苷酸转移标记法进一步证实茶多酚可诱导HepG-II细胞的凋亡。提示茶多酚可诱导体外培养的肝癌细胞株HepG-II细胞发生凋亡,具有抗肝癌的作用。     

放射诱导大鼠脑神经元和胶质细胞凋亡及敏感性的比较研究

目的　探讨X射线照射对大鼠脑神经元和胶质细胞凋亡的影响。方法　大白鼠分对照组及 2 ,4 ,6 ,8GyX射线照射组 ,照射后 1,2 ,4 ,6 ,12 ,2 4h分别取材进行光镜、电镜及DNA电泳观察 ,并用双标法计数凋亡的神经元数、胶质细胞数。结果　照射后鼠脑细胞系 2种细胞均产生凋亡的改变 ,胶质细胞凋亡率较神经元升高显著 (P <0 0 0 0 1) ,随着剂量的增高细胞凋亡率逐渐增多。结论　X射线能诱导鼠脑神经元和胶质细胞凋亡 ,凋亡率有剂量依赖性和时间规律性 ,其中胶质细胞最敏感。     

Dose-response for radiation-induced apoptosis, residual 53BP1 foci and DNA-loop relaxation in human lymphocytes

The purpose was to compare the radiation-induced apoptosis in human lymphocytes with DNA-loop relaxation and DNA damage as a function of radiation dose and time after exposure. Morphological changes were analysed by staining with fluorescent dyes and apoptotic fragmentation of DNA with conventional agarose gel electrophoresis, pulsed-field gel electrophoresis (PFGE) and alkaline comet assay. Viability was estimated by trypan blue assay. The levels of protein p53 (TP53) were determined with Western blot. Relaxation of DNA-loops was analysed by the method of anomalous viscosity time dependence (AVTD) and neutral comet assay. Induction and repair of double-strand breaks (DSB) was studied by PFGE and by immunostaining of the TP53 binding protein 1 (53BP1). At various time points of apoptosis, there was a linear dose dependence for all apoptotic end-points up to 1-2 Gy followed by a plateau at higher doses. Immediately after irradiation, relaxation of DNA-loops due to strand breaks was observed. This relaxation had a similar dose-response with saturation at 2-3 Gy. This dose induced approximately one single-strand break (SSB) per 2 Mb of DNA, a value close to the average size of DNA-loops in resting lymphocytes. Similar saturations in dose-responses for apoptosis and DNA-loop relaxation were also observed if cells were treated by camptothecin (CPT) or etoposide VP-16, drugs that relax DNA-loops by induction of SSB and DSB, respectively. The PFGE data showed that the vast majority of DSB were repaired within few hours after irradiation. However, approximately 1.4 foci/Gy/cell, that corresponded to around 3.5% of initial DSB, remained in cells even 24 h after irradiation as measured with immunostaining. The probability to produce one or more than one residual foci per cell was calculated. Radiation at 2-3 Gy induced at least one residual 53BP1 focus per cell. The dose-responses for DNA-loop relaxation, induction of at least one residual 53BP1 foci per cell and apoptosis saturated at 2-3 Gy. The correlation between dose-responses obtained suggested that the DSB in residual foci and relaxation of DNA-loops may be linked to induction of radiation-induced apoptosis in lymphocytes.     

Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells

Purpose : To examine how hypoxia influences ionizing irradiation-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. Materials and methods : Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. Results : In HL60 cells, apototic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-strand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. Conclusion : These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.     

快中子诱导人鼻咽癌细胞系凋亡的研究

目的　研究中子不同剂量照射人鼻咽癌（ＣＮＥ２） 细胞凋亡发生特点及与Ｘ射线所致凋亡的差异。探讨凋亡在中子治疗肿瘤中的作用及临床意义。方法　采用琼脂糖凝胶电泳及ＤＮＡ特异性荧光染色方法（Ｈｏｅｃｈｓｔ３３３４２） 检测照射后不同时间人鼻咽癌（ＣＮＥ２）细胞。结果　发现中子可诱导人鼻咽癌细胞发生凋亡,这种凋亡发生存在着一定的时间剂量相关性。在相同剂量照射下,同一时间点上,中子照射所致的凋亡反应强于Ｘ 射线所致的凋亡。结论　快中子照射离体细胞可引起较强的凋亡反应。中子杀伤肿瘤的机理可能也是主要通过凋亡途径来实现的。     

Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells

PURPOSE: To examine how hypoxia influences ionizing irradiation-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. MATERIALS AND METHODS: Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. RESULTS: In HL60 cells, apototic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-strand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. CONCLUSION: These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.