碱性单细胞凝胶电泳预测肿瘤细胞内在放射敏感性研究

应用克隆形成法和碱性单细胞凝胶电泳技术检测辐射诱导的人红白血病细胞株K562、人结肠腺癌细胞株LS-T-117和鼠胶质瘤细胞株C6的初始DNA单链断裂数及单链断裂后的修复与细胞内在放射敏感性之间的关系。结果表明,3种细胞系的放射敏感性依次为K562>LS-T-117>C6;3种细胞系的DNA迁移距离都随着照射剂量的增加而增大,呈良好的剂量-效应关系。在相同剂量下,辐射诱导的DNA单链的初始断裂数目也依次为K562>LS-T-117>C6;3种细胞系经10Gy X射线照射并在PBS中培养不同时间后DNA迁移距离都有较大幅度的下降,但下降幅度依次为C6>LS-T-117>K562,在相同剂量下辐射诱导的DNA单链断裂后的修复能力也依次为C6>LS-T-117> K562。结果显示,辐射诱导的DNA单链断裂及修复与细胞内在放射敏感性有很好的相关性,可用于人体肿瘤细胞内在放射敏感性的预测。     

用HPRT基因突变及CBMN研究AT细胞高辐射敏感性

研究了毛细血管扩张性共济失调症(Ataxia?telangiectasia,AT)患者皮肤的成纤维细胞系AT5BIVA(AT细胞)的高辐射敏感性。以源于正常人皮肤的成纤维细胞系GM0639(GM细胞)为对照,利用次黄嘌呤磷酸核糖转移酶基因(Hypoxanthinephospho?ribosyltransferase,hprt)位点突变分析技术及胞质分裂阻滞微核法(Cytokinesis?Blockmicronucleusmethod,CBMN),在AT细胞和GM细胞经Coγ射线0、1、2、3、4Gy60照射后,观察比较AT细胞和GM细胞之间hprt基因位点突变频率(hprtMF)、微核率(MNF)及微核细胞率(MNCF)的差异,并分别进行曲线拟合。在各剂量点,AT细胞hprt基因突变频率、微核率及微核细胞率均明显高于GM细胞,其差别具有显著性统计意义(p<0.01);AT和GM细胞hprt基因突变频率、微核率及微核细胞率均与照射剂量呈正相关,可拟合成剂量效应直线方程y=a bx。结果表明,毛细血管扩张性共济失调症患者AT细胞辐射敏感性显著高于GM细胞,具有高辐射敏感性。     

60Coγ射线对AT细胞SOD活性和MDA含量的影响研究

研究毛细血管扩张性共济失调症（AT）患者皮肤的成纤维细胞系AT5BIVA（AT细胞）的应对电离辐射导致细胞氧化损伤的能力。以源于正常人皮肤的成纤维细胞系GM063（GM细胞）为对照,采用超氧化物歧化酶（Superoxide dismutase,SOD）、丙二醛（Malondialdehydc,MDA）测定试剂盒,AT细胞和GM细胞经60^Coγ射线0、1、2、3、4Gy照射后,观察比较它们的SOD活性和MDA含量的差异,并分别进行曲线拟合。结果显示在1、2、3、4Gy剂量照射下,AT和GM细胞中SOD活性均随受照剂量增加而明显降低（P〈0．01）,同一剂量点的AT细胞中SOD活性明显低于GM细胞（P〈0．05）;AT和GM细胞中MDA含量均随受照剂量增加而升高（P〈0．05）,同一剂量点的AT细胞中MDA含量明显高于GM细胞（P〈0．05）。AT细胞和GM细胞的SOD活性和MDA含量均与照射剂量呈线性关系。结果表明由于AT细胞中SOD活性降低,导致AT细胞应对辐射的氧化损伤能力不足,引起AT细胞中MDA含量增加,是AT细胞高辐射敏感性的原因之一。     

60Co γ射线照射对AT和GM细胞增殖能力及O2.-浓度的影响

为探求AT5BIVA细胞辐射敏感性与超氧阴离子自由基O2.-之间的联系,以正常人皮肤成纤维细胞GM0639为对照,用3H-TdR掺入法和细胞色素C还原法分别测定正常及照射条件下细胞增殖能力及其培养介质中O2.-浓度(nmol/106细胞).结果表明,在正常培养条件下,两种细胞的增殖能力和向培养介质中释放O2.-的量均随培养时间的延长而增加.在不同剂量60Co γ射线照射条件下,两种细胞的增殖能力和向培养介质中释放O2.-的量均随剂量增大而减小;两种细胞的增殖能力均与其培养介质中O2.-浓度密切相关,且AT5BIVA细胞的增殖速率大于GM0639细胞.提示AT5BIVA细胞高辐射敏感性与O2.-产生量密切相关.     

小鼠乳癌细胞辐射敏感突变株SX-9DNA双链断裂及其重接修复研究

本研究采用一种新的细胞DNA双链断裂检测方法——脉冲电场电泳法,检测了两株来源相同而辐射敏感性不同的细胞SR-1和SX-9的X线照射所致DNA双链断裂的产生和修复。结果表明两株细胞的DNA双链断裂产生没有差别,而辐射敏感细胞SX-9的DNA双链断裂重接修复能力明显低于SR-1细胞,本文对此结果与细胞辐射敏感性的关系进行了讨论。     

电离辐射引起的DNA链断裂损伤及其修复

DNA链断裂是电离辐射的主要生物学效应之一,单链断裂由一次击中造成,所以与辐射剂量成线性关系,双链断裂可以一次击中造成,也可由两个位置相关的单链断裂迭加而成,所以与辐射剂量成线性——平方关系。已有各种理化方法能测定活细胞内的单链断裂和双链断裂数。细胞具有修复DNA链断裂的能力,但其机制远不如切除修复机制那么清楚。未修复的链断裂是致死的,被修复的单链断裂完全恢复DNA的正常结构与功能,被修复的双链断裂的生物学效应如何还不清楚。剂量率对辐射的生物学效应有重要影响。     

病毒蛋白与宿主相互作用及其对细胞放射敏感性的影响

为了解病毒感染对电离辐射生物效应的可能影响和作用机理,本文着重评述了病毒编码蛋白与宿主细胞蛋白的相互作用,特别是干扰宿主DNA 损伤修复、影响辐射致细胞凋亡、干扰DNA损伤后的细胞周期检查点,以及其对哺乳动物细胞的放射敏感性的影响.病毒蛋白与宿主细胞的相互作用,不仅在病毒感染及相应致病作用中扮演着重要角色,而且也影响到被感染组织对辐射或某些化学物质的反应.业已证实,一系列的病毒蛋白与宿主蛋白发生作用,这种作用与包括DNA修复、凋亡、细胞周期检查点在内的DNA双链断裂的细胞反应有关,造成宿主蛋白的细胞定位改变、降解、失活或激活等;另外一些蛋白直接或间接调节多种宿主基因的转录水平,或诱发辐射损伤反应相关蛋白的转录后修饰的变化,使得这些基因产物上调或下调、活性改变.病毒蛋白与宿主的相互作用,在一定程度上改变了细胞的放射敏感性.充分、准确地了解病毒感染的辐射生物学意义,将促进我们关于人体对辐射健康效应的个体敏感性的理解和对未来辐射防护思路的拓展.     

TGF-β1抑制剂增加H460肺癌细胞的放射敏感性

探索一种TGF-β1受体激酶的选择性抑制剂SB431542对H460非小细胞肺癌细胞的放射增敏作用及对其DNA损伤应答体系的干扰。采用克隆形成实验检测SB431542对H460细胞放射敏感性的影响;用移植瘤实验验证SB431542在体内对H460细胞的放射增敏作用;采用流式细胞术检测细胞周期分布和细胞凋亡;用免疫荧光检测53BP1 foci和磷酸化的DNA-PKcs foci。结果发现,SB431542可增加H460的放射敏感性。照射前用SB431542预处理H460细胞虽然快速启动了细胞的DNA损伤应答反应,但却抑制了DNA双链断裂的非同源末端链接修复,因此,导致细胞残留更多未修复的DNA双链断裂,从而产生更严重的细胞周期阻滞。研究还发现,这种增敏作用与细胞凋亡无关。体内移植瘤实验表明,与单独照射组相比,连续3次用SB431542联合5 Gy的X-射线处理明显在处理后第5~12 d之间降低了肿瘤的生长。这些结果显示,在照射前用SB431542抑制肿瘤细胞的TGF-β1通路,能降低其DNA损伤修复能力,改变细胞周期分布,降低细胞克隆形成能力,延缓肿瘤的生长,因此用SB431542抑制TGF-β1通路可作为一些类型肺癌病人放疗的有效辅助手段。     

DNA损伤修复能力对辐射适应性反应的影响

采用野生型的中国仓鼠卵母细胞(CHO-9)及其DNA损伤修复缺陷型细胞:EM-C11(DNA单链断裂修复缺陷)和XR-C1(DNA双链断裂修复缺陷),首先进行低剂量(0.016 Gy或0.08 Gy)的初始照射,间隔4 h或7 h后再进行高剂量(1 Gy)的攻击照射,然后检测细胞微核形成率和克隆形成率的变化.结果显示:当初始剂量为0.08 Gy,间隔4 h后再施以1 Gy的攻击照射时,三株细胞只有野生型的CHO-9有辐射适应性反应产生;但当间隔时间延长到7 h时,CHO-9和EM-C11均产生了辐射适应性反应.当把初始照射剂量降低为0.016 Gy,间隔4 h再施以1 Gy的攻击照射时,三株细胞均产生了辐射适应性反应.表明辐射适应性反应不仅与初始照射剂量及间隔时间有关,还与DNA损伤修复密切相关.     

微细胞介导染色体转移法提高AT5 BIVA细胞对电离辐射的抗性

ＡＴ５ＢＩＶＡ细胞是一株经ＳＶ４０病毒转化的ＡＴ病人皮肤成纤维细胞，对γ射线高度敏感。实验用ＦＤ３（含人第１１号染色体的人鼠杂种细胞）、ＦＤ８（不含人第１１号染色体的人鼠杂种细胞）、ＬＭ／ＴＫ鼠细胞）为洪体，通过微细胞介导染色体转移（ＭＭＣＴ）向ＨＴ５ＢＩＶＡ细胞导入人或鼠的完整染色体，经两次３Ｇｙγ射线照射筛选后，获得ＡＴ５ＢＩＶＡ与ＦＤ３微细胞融合的杂合细胞ＡＴ／ＦＤ３－１，对γ射线抗性有显著提高。而ＦＤ８或ＬＭ／ＴＫ的微细胞与ＡＴ５ＢＩＶＡ细胞的杂合细胞，对γ射线抗性未增加。枝型分析表明ＡＴ／ＦＤ３—１细胞中包含了来源于ＦＤ３细胞的人第１１，１４号染色体和数条鼠染色体。通过对照实验，排除了人１４号和鼠染色体提高ＡＴ／ＦＤ３－１细胞对γ射线抗性的可能性．确认人第１１号染色体与ＡＴ细胞对电离辐射敏感性相关，提示人第１１号染色体上可能存在决定细胞对γ射线抗性的相关基因。     

Evaluation of radiosensitivity of human tumor cells after irradiation of γ-rays based on G2-chromosome aberrations

The aim of the present investigation is to determine initial G2-chromosome aberrations and to validate whether the G2-chromosome aberrations can predict the cellular clonogenic survival in human tumor cell lines. Cell lines of human ovary carcinoma cells (HO8910) and human hepatoma cells (HepG2) were irradiated with a range of doses and assessed both for initial G2-chromosome aberrations and for cell survival after γ-irradiation. The initial G2-chromosome aberrations were measured by counting the number of G2-chromatid breaks after irradiation, detected by the premature chromosome condensation technique, and the G2-assay method. Cell survival was documented by a colony formation assay. A linear-quadratic survival curve was observed in both cell lines. The dose-response results show that the numbers of G2-chromatid breaks increase with the increase in dose in the two cell lines. At higher doses (higher than 4 Gy) of irradiation, the number of G2-chromatid breaks for the G2-assay method cannot be determined because too few cells reach mitosis, and hence their detection is difficult. A good correlation is found between the clonogenic survival and the radiation-induced initial G2-chromatid breaks per cell (r=0.9616). The present results suggest that the premature chromosome condensation technique may be useful for determining chromatid breaks in G2 cells, and the number of initial G2-chromatid breaks holds promise for predicting the radiosensitivity of tumor cells.     

Evaluation of radiosensitivity of human tumor cells after irradiation of γ-rays based on G2-chromosome aberrations

1 Introduction It is well established that the radiosensitivity is dependent on cell-cycle progression, and among stages of interphase, G2 phase is the most radiosensitive fol- lowed by G1 phase, and S phase, which is the least ra- diosensitive, whereas G0 phase is radioresistant under conditions of oxygen deprivation and sufficient repair time [1-3]. Previous studies have concluded that the G2 phase is a very important stage. Some researchers have studied the chromosome aberrations in G2 ph…     

AT细胞特征及纠正细胞基因缺陷的研究

实验证明一株经SV 40病毒转化的毛细血管扩张性共济失调症病人的皮肤成纤维细胞AT 5 BIVA,对电离辐射和博莱霉素高度敏感,对γ射线诱发的DNA合成抑制呈抗性。本文尝试用DNA介导基因转移法向AT细胞导入人基因组DNA或其酶切片段,未能获得稳定的对γ射线抗性的转化细胞。染色体核型分析证实AT 5 BIVA细胞的第11号染色体中的一条缺失或异常。本文对外源目的基因未能稳定植入AT细胞的原因进行了讨论。     

Relationship between cellular radio-sensitivity and naked DNA damage in mammalian cells exposed to heavy ions

The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by ~(12)C ions irradiation was examined in four kinds of cells, Melanoma B16, cervical squamous carcinoma HeLa, Chinese hamster V79 and hepatoma SMMC-7721. Cell survival was determined by a colonogenic assay, and the sensitivity was described by D_(50)(the dose of radiation necessary to reduce the survival to 50%). For all cell lines, D_(50) ranged from 0.74 Gy to 3.85 Gy, among them B16 was the most radiosensitive to ~(12)C ions, and V79 and HeLa cells had almost the same radio-sensitivity, SMMC-7721 was the last. The induction of deproteinized DNA double-strand breaks induced by ~(12)C ions were measured by pulsed-field gel electrophoresis (PFGE), and the initial yield of the deproteinized DNA dsbs per unit dose was expressed as the DNA double break level (L). A linear dose-response curve was seen for initial DNA dsbs for all cell lines (slopes range from 0.40-0.98 (DSBs/100Mbp/Gy)). V79 was the steepest, B16 was the last.There was an inverse relationship between the initial DNA dsb and D_(50) if the B16 cell line was not considered, but there was no relativity even excludes the B16 cell line. The present results indicate that there is no relationship between cellular sensitivity and initial DNA dsb, even exclude the effects of chromatin structure.     

用染色体畸变分析对比两种病理类型鼻咽癌细胞株的辐射敏感性

用染色体畸变分析，检测两种病理类型鼻咽癌细胞株的放射敏感性。证实来源于高分化鼻咽癌病人鼻咽部肿瘤的细胞株，其辐射敏感性较低分化型细胞株明显减低，本结果可提供临床治疗时参考。     

NASOPHARYNGEAL CARCINOMA RADIOSENSITIVITY PREDICTION BY CYTOKINESIS－BLOCK MICRONUCLEUS ASSAY

ＮＡＳＯＰＨＡＲＹＮＧＥＡＬ　ＣＡＲＣＩＮＯＭＡ　ＲＡＤＩＯＳＥＮＳＩＴＩＶＩＴＹ　ＰＲＥＤＩＣＴＩＯＮ　ＢＹ　ＣＹＴＯＫＩＮＥＳＩＳ－ＢＬＯＣＫ　ＭＩＣＲＯＮＵＣＬＥＵＳ　ＡＳＳＡＹＹａｎｇＸｉｎｇ（杨星）；ＳｈｉＪｉａｎｈｕｉ（史剑慧）ａｎｄＣｈ...     

Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

In this paper, we study the ability of DNA-PK-deficient (M059J) and -proficient (M059K) cells to undergo the rate of cellular proliferation, cell cycle distribution and apoptosis after 10 Gy X-ray irradiation, and the role of DNA-PK in radiosensitivity. The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells. A strong G2 phase arrest was observed in M059J cells post irradiation. Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h. Altogether, the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage, a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint, by which DNA-PK may affect cellular sensitivity to ionizing radiation.