肿瘤细胞对p(35)Be快中子的辐射敏感性研究

目的 比较肿瘤细胞p(35)Be块中子及γ射线的辐射敏感性,为肿瘤的快中子治疗提供理论依据。方法 用细胞集落在存活方法研究人黑色素瘤细胞（WM9839）、人口 皮癌细胞（KB）、人结肠腺癌细胞（LS－T－117）和人前列腺癌细胞（PC3M）等4种细胞对快中子及γ射线的辐射敏感性,用彗星电泳技术研究WM9839细胞在快中子及γ射线照射后DNA损伤的修复效应。结果 细胞存活实验表明,p(35)Be快中子照射后4种肿瘤细胞的D0值（或SF2值）较γ射线照射后差异减小,即4种肿瘤细胞对快中子的辐射敏感性差异减小;快中子2Gy照射后,WM9839细胞DNA损伤修复曲线整体上下降较γ射线2Gy照射后慢,到180min时,DNA损伤残留率明显高于γ射线2Gy照射。结论 快中子治疗肿瘤可以很好地弥补低LET射线放射治疗的不足,特别是对低LET射线较为耐受的肿瘤细胞,如KB细胞和WM98309细胞。     

60Coγ射线对hOGG1低表达细胞株放射敏感性的影响

目的研究^60 Co γ射线对DNA碱基切除修复基因hOGG1低表达细胞株放射敏感性的影响.方法以A549细胞和通过稳定转染hOGG1核酶而获得的hOGG1低表达的A549-R细胞为研究对象,用MTT法测定不同剂量γ射线照射后两种细胞的存活率;单细胞凝胶电泳检测^60 Coγ射线处理后两种细胞DNA损伤与修复的差异;流式细胞术检测照射后两种细胞的周期分布、凋亡率和细胞增殖指数.结果照射后两种细胞的存活率均随照射剂量的增加而下降,但A549-R细胞组的存活率显著低于相同剂量组的A549细胞（P＜0.05）;所设剂量均可诱导细胞DNA损伤,但DNA迁移长度和彗星细胞率在两种细胞间差异无统计学意义（P＞0.05）;损伤后的修复在两种细胞间差异有统计学意义（P＜0.05）,A549-R细胞的修复能力远远低于A549细胞.流式细胞术检测结果表明：照射后两种细胞都表现为G0/G1期细胞阻滞,细胞凋亡随照射剂量的增加而增加,细胞增殖指数随照射剂量的增加而降低,这些变化均以A549-R细胞更为明显.结论hOGG1低表达使得细胞DNA修复能力降低,细胞周期阻滞于G0/G1期、细胞增殖减慢、凋亡增加、存活率下降,从而使细胞对^60Coγ射线的放射敏感性增强.     

应用脉冲电场凝胶电泳分析X射线诱发人骨肉瘤细胞DNA双链断裂与损伤修复效应

目的 研究电离辐射诱发人骨肉瘤肿瘤细胞DNA双链断裂与辐射损伤修复效应, 观察辐射损伤、损伤修复效应与肿瘤细胞辐射敏感性之间的关系。方法 选用强制均匀电场电泳, 分别测定经不同剂量X射线照射和相同剂量照射后培养不同时间, 人骨肉瘤Rho0和143.B肿瘤细胞株DNA双链断裂。结果 (1)X射线诱发人骨肉瘤肿瘤细胞的DNA双链断裂与辐射剂量呈线性正比关系; (2)培养后的人骨肉瘤肿瘤细胞对辐射诱发的DNA双链断裂具有一定修复能力; (3)Rho0比143.B细胞株具有更高的辐射敏感性; (4)脉冲电场凝胶电泳技术是分析人肿瘤细胞DNA双链断裂的敏感方法。结论 脉冲电场凝胶电泳是分析人肿瘤细胞DNA双链断裂的敏感方法; 电离辐射诱发人骨肉瘤细胞DNA双链断裂与损伤修复效应和肿瘤细胞的辐射敏感性有密切关系。     

Tip60基因沉默对细胞DNA双链断裂修复和辐射敏感性的影响

目的 探讨Tip60对细胞辐射敏感性的影响及相关机制。方法 采用siRNA和Tip60乙酰转移酶抑制剂漆树酸,抑制U2OS细胞中Tip60的表达或乙酰转移酶活性;用克隆形成率分析细胞对⁶⁰Co γ射线的敏感性;采用γ-H2AX原位免疫荧光集簇点法,检测DNA双链断裂损伤修复;用免疫共沉淀检测蛋白质的相互作用。结果 siRNA沉默Tip60表达明显提高了U2OS细胞对1、2 Gy中、低剂量γ射线的敏感性(t=3.364、3.979,P＜0.05),但对4 Gy大剂量照射的细胞存活率无明显影响。γ-H2AX集簇点检测结果表明,照射后1、4和8 h,Tip60失活导致细胞DNA双链断裂修复能力降低(t=3.875、3.183和3.175, P<0.05)。细胞在受到电离辐射损伤后,Tip60与DNA修复蛋白DNA-PKcs发生相互作用,漆树酸能抑制DNA-PKcs的T2609位点的磷酸化。结论 Tip60通过与DNA-PKcs相互作用,调控细胞DNA双链断裂修复机制,对细胞辐射敏感性产生影响。     

线粒体DNA4977bp缺失和彗星分析评价肿瘤细胞的辐射敏感性

目的 探讨mtDNA4977bp缺失和彗星分析用于肿瘤细胞辐射敏感性检测的可行性。方法 选取3种不同肿瘤细胞株:肝癌细胞(HepG₂)、食管癌细胞(EC-9706)和乳腺癌细胞(MCF-7)。采用MTT法检测肿瘤细胞经γ射线照射后的存活分数(SF);巢式PCR法测肿瘤细胞的mtDNA4977bp缺失率;单细胞凝胶电泳(SCGE)检测肿瘤细胞的DNA断裂水平。结果 用MTT法发现HepG₂ 和EC-9706细胞的辐射敏感性高于MCF-7细胞。8 Gy照射后HepG₂和EC-9706 mtDNA4977bp缺失率明显高于MCF-7细胞(P<0.05),证实HepG₂和EC-9706细胞的辐射敏感性高于MCF-7细胞。多种方法分析结果说明3种肿瘤细胞在8Gy照后表现出的辐射敏感性差异有统计学意义。结论 多种生物学指标的综合应用,可能更加客观准确地评价肿瘤细胞的辐射敏感性。     

60Coγ射线诱导EJ细胞DNA损伤及γH2AX 表达的研究

目的 探讨不同剂量⁶⁰Coγ射线对EJ细胞DNA损伤的情况,不同剂量⁶⁰Coγ 射线照射EJ细胞后诱导磷酸化组蛋白H2AX焦点形成,以及与γH2AX表达量的关系。方法 单细胞凝胶电泳检测DNA链断裂损伤情况。免疫荧光法检测不同剂量γ 射线照射后立即、以及2 Gy γ 射线照射后不同时间的EJ细胞中γH2AX焦点的数量。流式细胞分析法检测不同剂量γ 射线照射后EJ细胞中γH2AX 蛋白表达量的变化。结果 单细胞凝胶电泳结果显示,γ射线照射后DNA损伤情况明显加重,随照射剂量的增加,细胞尾矩不断加大,0 Gy组尾矩为0.24,4 Gy照射组尾矩为5.26;免疫荧光结果显示,随着照射剂量的增加,γH2AX焦点数目及大小均明显增加,照射的剂量范围从0.1~4 Gy均可检测,且照射剂量和焦点形成数目之间存在剂量-效应关系,0.1 Gy照射组每个细胞中的焦点数平均达12.37个,4 Gy照射组每个细胞中的焦点数平均达46个;2 Gy γ 射线照射后24 h仍可检测到γH2AX焦点,随时间延长焦点数目减少、强度减弱,具有时间依赖性。流式细胞检测结果表明,γ 射线照射后γH2AX 蛋白表达量明显增加,呈现明显量效关系,0.1和4 Gy照射组γH2AX阳性细胞表达率分别为7.4%和29.2%。结论 免疫荧光法检测照射后γH2AX焦点数目比其他实验方法更能敏感、直观的反映DNA损伤及修复情况,有望成为检测辐射损伤的理想生物指标。     

p~(53)依赖途径的辐射诱导潜在致死损伤的研究

目的 :研究潜在致死损伤 (PL D)是否与 p5 3有关。方法 :采用 12种肿瘤细胞株和 3种人二倍体纤维原细胞株分析辐射敏感性、DNA双链断裂重连接、PL D表达及修复 (PL DR)。其中 7种细胞株 p5 3正常表达 ,8种 p5 3功能缺陷 (6种基因突变 ,2种 HPV 16 E6转染 p5 3表达正常的细胞株 )。取指数生长期 (辐射前低密度培养 18h)和高峰生长期细胞 (待细胞长满后继续培养 3 d以上 ) ,1 3 7　 Csγ射线或 X射线 (剂量率为 1Gy/ min,剂量为 7Gy)照射后立即或于 2 4h以后消化成单细胞悬液 ,培养 2～ 3周 ,固定 ,0 .2 5 %结晶紫染色 ,观察细胞克隆…     

淋巴细胞DNA双链断裂用于放射损伤检测的可行性研究

目的探讨受照后淋巴细胞DNA双链断裂和凋亡作为放射损伤生物学检测指标的可行性。方法采用DH值中性彗星电泳方法，检测人外周血离体照射0．1～4Gy后6h和24h淋巴细胞彗星率和彗星尾长，并观察其量效关系。结果照后6h和24h彗星率呈剂量依赖性上升，反应剂量阈值分别为0．6Gy和0．1Gy，γ分别为0．967和0．927；照后6h彗星尾长亦呈剂量依赖性上升，γ为0．847。结论中性彗星电泳法结果提示在照射剂量和淋巴细胞彗星率及彗星尾长间存在较紧密的相关性。     

DNA双链断裂残留损伤与癌细胞辐射敏感性研究

目的 了解不同组织来源癌细胞株和人体肿瘤组织原代细胞的DNA双链断裂损伤修复的个体差异性,探寻预测癌细胞辐射敏感性的生物指标。方法 ⁶⁰Co γ射线照射诱发DNA损伤,脉冲电场凝胶电泳检测DNA双链断裂损伤修复,细胞克隆形成能力法检测细胞辐射敏感性。结果 8个不同组织来源癌细胞株的辐射敏感性有较大的差异(D₀为0.65~2.15 Gy),不同细胞株20 Gy γ射线照射诱发产生的DNA双链断裂原初损伤有一定的差别,但与细胞辐射抗性无相关性。辐射敏感细胞SX-10的DNA双链断裂修复缺陷发生在早期快速修复相,而A2780细胞的修复缺陷是发生在晚期慢速修复相。20 Gy照射修复2 h后DNA双链断裂残留量与细胞辐射敏感性指标D₀或SF₂值有显著的相关性。不同个体患者脑肿瘤组织原代细胞之间,辐射诱发DNA双链断裂的修复反应存在明显差异,修复2 h后残留损伤的个体差异性分布类似于癌细胞株。结论 DNA双链断裂残留损伤与癌细胞辐射抗性有显著相关性,可作生物指标预测肿瘤组织细胞对放射治疗的反应性。     

异染色质蛋白HP1α参与辐射损伤修复的研究

目的 研究HP1α对辐射损伤修复的影响。方法 用反义技术单独抑制HP1α和联合抑制HR24L表达，观察细胞对辐射敏感性的影响，用免疫共沉淀法观察HP1α是否参与修复蛋白复合物的形成。结果 单独抑制HP1α，细胞对辐射敏感性与正常细胞相比差异无显著性，联合抑制HP1α和HR24L时，细胞对辐射的敏感性比单独抑制HR24L时显著增加；20 Gyγ射线照射后8h，可检测出HR24L与HP1α复合物的存在。结论 HP1α与修复蛋白相互作用，参与DNA损伤修复，从而影响细胞对辐射的敏感性。     

应用脉冲电场凝胶电泳分析X射线诱发人骨肉瘤细？…

目的 研究电离辐射诱发人骨肉瘤肿瘤细胞ＤＮＡ双链断裂与辐射损伤修复效应,观察辐射损伤、损伤修复效应敏感性之间的关系。方法 选用强制均匀电场电泳,分别测定经不同的剂量Ｘ射线照射和相同剂量照射后培养不同时间,人骨肉瘤Ｒｈｏ０和１４３．Ｂ肿瘤细胞株ＤＮＡ双链断裂。结果 （１）Ｘ射线诱发人骨肉瘤瘤细胞的ＤＮＡ双链辐射剂量呈线性正比关系;（２）培养后的人骨肉瘤肿瘤细胞对射诱发的ＤＮＡ双链断裂具有一定修复能力     

辐射诱导人细胞系CEM、外周血单个核细胞DNA修复基因hHR21~(sp)的表达研究

目的　研究辐射对人T淋巴细胞白血病细胞系 (CEM)、外周血单个核细胞的hHR2 1sp基因转录表达水平的影响及意义。方法　分别对人T淋巴细胞白血病细胞系CEM和正常人外周血单核细胞在UV或γ辐射后不同时间提取细胞总RNA ,通过RT PCR与hHR2 1sp 基因特异引物杂交 ,以 β actin为内参照放射影像密度扫描检测人T淋巴细胞白血病细胞系CEM、单核细胞DNA修复基因表达。结果　在UV 辐射、γ 辐射后早期 (3～ 6h) ,人T淋巴细胞白血病细胞系CEM和淋巴细胞hHR2 1sp基因的表达水平明显增加 ,照射后 6hhHR2 1sp基因的表达水平增加最多且UV辐射更明显 ,在晚期 (9h)降低。比较人细胞系CEM和淋巴细胞两者hHR2 1sp基因的表达水平 ,γ辐射 (3Gy)后淋巴细胞对hHR2 1sp基因表达高于人细胞系CEM ,且表达增加时间较长 ,达 9h ;而人细胞系CEM受到γ辐射后早期表达增加 ,在 6～ 9h后表达降低。结论　人T淋巴细胞白血病细胞系 (CEM)和人淋巴细胞的DNA修复基因hHR2 1sp基因在一定剂量辐射 (UV、γ辐射 )范围内其表达水平随辐射剂量增加而诱导表达水平增高 ,且对UV辐射更敏感 ;提示hHR2 1sp基因在人细胞系CEM细胞和人单核细胞在照射损伤后表达增加 ,可能是促进单核细胞损伤修复的原因之一。     

Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal,NBS and AT fibroblasts

Purpose : To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay. Materials and methods : Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay. Results : The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7 ±0.2 to 5.0 ±0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks. Conclusions : The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.     

Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal, NBS and AT fibroblasts

PURPOSE: To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay. MATERIALS AND METHODS: Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay. RESULTS: The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7+/-0.2 to 5.0+/-0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks. CONCLUSIONS: The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.     

Optimization and Standardization of the “Comet Assay” for Analyzing the Repair of DNA Damage in Cells

BACKGROUND: The "comet assay" has become an interesting and a very useful tool for the analysis of the induction and amount of DNA damage in single cells thus offering the opportunity to measure the effectiveness of DNA repair. On the basis of the Ostling and Johanson protocol we have developed a modified method with increased sensitivity and high reproducibility. MATERIAL AND METHODS: Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique. RESULTS: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for "comet" analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. CONCLUSIONS: This paper presents data on the optimization and standardization of the original "comet assay" leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the "comet assay" may have the potential for the estimation of individual radiosensitivity.     

电离辐射对不同肿瘤细胞细胞周期的影响

目的 研究电离辐射对不同肿瘤细胞细胞周期的影响为肿瘤放疗及化疗提供科学依据。方法 处于细胞周期各时相的细胞百分数采用流式细胞术进行检测。结果 研究表明：电离辐射作用后,ＨｅｌａＳ３和Ｓ１８０细胞发生了Ｓ和Ｇ２期阻滞,而ＤＬ－４细胞则发生Ｇ１和Ｇ２期阻滞,Ｂ１６各时相细胞数无列出较高的辐射抗性。结论 电离辐射作用后,不同肿瘤细胞的辐射抗性、即辐射敏感性有较大差异。其细胞周期的变化规律亦不相同。     

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.