低剂量辐射诱导适应性反应的分子机制研究现状

低剂量辐射(low dose radiation，LDR)可以增强细胞对随后进行的攻击性剂量(challenge dose)照射的抵抗能力，从而降低攻击性照射引起的染色体畸变和DNA损伤。人们把LDR的这种效应称之为“低剂量辐射诱导的适应性反应”。低剂量辐射诱导适应性反应的分子机制主要涉及细胞信号转导、ROS(活性氧物质)的作用和DNA修复兴奋效应等方面。     

低剂量辐射的细胞遗传适应性反应机理研究现状

很多学者对LDR(低剂量辐射)可以在体内及体外培养的细胞中诱导出适应性反应的机理进行了深入的研究,发现适应性反应的诱导与很多因素有关,这些因素包括LDR诱导的DNA修复系统的激活、LDR诱导的基因和蛋白的作用、抗氧化物酶的作用、细胞信号转导以及与p53蛋白有关的细胞周期阻滞的影响等。     

低剂量辐射诱导适应性反应的分子机制研究现状

低剂量辐射(low dose radiation,LDR)可以增强细胞对随后进行的攻击性剂量(challenge dose)照射的抵抗能力,从而降低攻击性照射引起的染色体畸变和DNA损伤。人们把LDR的这种效应称之为"低剂量辐射诱导的适应性反应"。低剂量辐射诱导适应性反应的分子机制主要涉及细胞信号转导、ROS(活性氧物质)的作用和DNA修复兴奋效应等方面。     

低剂量辐射的细胞遗传适应性反应机理研究现状

很多学对LDR（低剂量辐射）可以在体内及体外培养的细胞中诱导出适应性反应的机理进行了深入的研究，发现适应性反应的诱导与很多因素有关，这些因素包括LDR诱导的DNA修复系统的激活、LDR诱导的基因和蛋白的作用、抗氧化物酶的作用、细胞信号转导以及与p53蛋白有关的细胞周期阻滞的影响等。     

低剂量辐射诱导小鼠胸腺细胞及其亚组的适应性反应

目的研究低剂量辐射预先照射以及随后大剂量照射后小鼠胸腺细胞Ｔ细胞受体（ＴＣＲ）、ＣＤ３、ＣＤ４和ＣＤ８分子表达的变化。方法ＴＣＲ、ＣＤ３表达采用间接荧光流式细胞术检测,ＣＤ４、ＣＤ８表达采用双参数直接免疫荧光流式细胞术检测。结果实验结果表明：单纯１５ＧｙＸ射线全身照射后ＴＣＲ,ＣＤ３阳性细胞数以及ＣＤ４－Ｄ８－,ＣＤ４＋ＣＤ８＋,ＣＤ４＋ＣＤ８－和ＣＤ４－ＣＤ８＋细胞数明显减少,当１５ＧｙＸ射线全身照射前６小时预先照射００７５Ｇｙ时,可明显减轻其后１５Ｇｙ照射对ＴＣＲ＋,ＣＤ３＋,ＣＤ４＋ＣＤ８＋,ＣＤ４＋ＣＤ８－和ＣＤ４－ＣＤ８＋的损伤作用。表现为各亚组细胞数显著高于单纯１５Ｇｙ照射组。ＣＤ４－ＣＤ８－亚组的细胞数无明显变化。结论００７５ＧｙＸ射线全身照射能够诱导胸腺细胞ＴＣＲ＋,ＣＤ３＋,ＣＤ４＋ＣＤ８＋,ＣＤ４＋ＣＤ８－和ＣＤ４－ＣＤ８＋亚组细胞的适应性反应。     

低剂量辐射诱导免疫适应性反应的剂量效应

本实验采用Ｘ射线全身照射，观察辐射诱导免疫适应性反应的预照射剂量（Ｄ１剂量）及其后损伤性剂量（Ｄ２剂量）的剂量范围．结果表明：Ｄ１在２５ｍＧｙ～１００ｍＧｙ范围内（剂量率为１２．５ｍＧｙ／ｍｉｎ），Ｄ２在１．０～１．５Ｇｙ范围内（剂量率为０．３３Ｇｙ／ｍｉｎ），Ｄ１与Ｄ２的时间间隔６小时，可诱导昆明小鼠胸腺细胞自发增殖力及丝裂原（ＣｏｎＡ和ＬＰＳ）刺激的脾细胞增殖反应等免疫适应性反应。     

低剂量辐射对细胞 DNA 修复兴奋效应

目的研究低剂量电离辐射对细胞ＤＮＡ双链断裂修复及基因突变适应性的兴奋效应，并探测辐射诱导ＤＮＡ结合蛋白。方法实验用小鼠ＳＲ－１细胞，６０Ｃｏγ射线照射；用６－巯基鸟嘌呤筛选ｈｐｒｔ基因突变克隆；脉冲电场凝胶电泳检测ＤＮＡ双链断裂；Ｓｏｕｔｈｗｅｓｔｅｒｎ印迹杂交探测ＤＮＡ结合蛋白。结果细胞受１ｃＧｙ预照射后１８小时、２４小时显著降低３Ｇｙ照射诱发的ｈｐｒｔ基因突变频率。预先受单次１ｃＧｙ照射刺激，或每天照射一次，连续１０天，显著增加３Ｇｙ照射细胞的ＤＮＡ双链断裂修复效率。１ｃＧｙ照射细胞后１６小时提取的核蛋白中，探测到损伤ＤＮＡ结合蛋白的诱导合成。结论低剂量辐射通过调节某些细胞辐射反应调控基因表达，诱导合成ＤＮＡ修复反应蛋白，增强细胞ＤＮＡ修复能力，减少基因突变的发生，提高细胞维持遗传稳定性机能。     

低剂量辐射诱导免疫适应性反应的最佳时间

本实验观察了低剂量Ｘ射线全身单次照射诱导昆明小鼠免疫适应性反应的最佳时间．证实当预照射剂量（Ｄ＿１剂量）为７５ｍＧｙ．剂量率１２．５ｍＧｙ／ｍｉｎ，损伤剂量（Ｄ＿２剂量）为１．５Ｇｙ，剂量率０．３３Ｇｙ／ｍｉｎ，Ｄ＿１与Ｄ＿２间隔６ｈ可诱导脾细胞对脂多糖反应的适应性反应，Ｄ＿１与Ｄ＿２间隔１２ｈ可诱导胸腺细胞自发增殖及脾细胞对ＣｏｎＡ及脂多糖反应的适应性反应．以上结果表明，当Ｄ＿２为１．５Ｇｙ时。７５ｍＧｙ诱导上述免疫适应性反应的最佳时间间隔为６ｈ及１２ｈ．     

Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity

Abstract

Purpose: A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity.

Materials and methods: Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632.

Results: When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1.

Conclusion: CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.     

ATM参与辐射损伤细胞学反应的研究

目的 克隆ＡＴＭ全长ｃＤＮＡ及含特异功能域的ｃＤＮＡ片段,寻找与ＡＴＭ相互作用的蛋白,分析ＡＴＭ在ＤＮＡ损伤名的分子机理。方法 利用长片段ＰＣ白话 增法,从人外周血来源的ｃＤＮＡ库中扩增ＡＴＭｃＤＮＡ;利用酵母双杂交系统筛选人外周血来源的ｃＤＮＡ库中与ＡＴＭ相互作用的蛋白。结果 经重叠ＰＣＲ扩增到ＡＴＭ全长ｃＤＮＡ,并筛选到数条与ＡＴＭＰ１３Ｋ激酶区相互作用的ｃＤＮＡ,并对其序列进行分析。结果 Ａ     

端粒与细胞DNA损伤反应

细胞的染色体DNA损伤监控和修复机制\和染色体末端的端粒保护机制是维护基因组稳定性的两大重要机制.这两个进化上高度保守的细胞生物学机制既有不同的分工和调控机制,又存在极为密切的内在联系.由于端粒和细胞DDR调控的异常与人类衰老和癌症有密切联系,因此对端粒与DDR系统的相互作用及协同性的深入研究,将对解决这些人类重大医学问题有重要影响.本文对端粒与细胞DNA损伤反应系统相互作用的研究进展进行综述.     

低剂量辐射诱导免疫适应性反应的实验研究

目的　研究低剂量辐射能否诱导免疫适应性应。方法　３ＨＴｄＲ 掺入法检测淋巴细胞增殖率;ＣＴＬＬ依赖株检测ＩＬ２ 活性;单克隆抗体免疫荧光标记,流式细胞术（ＦＣＭ） 检测淋巴细胞亚组及膜受体表达;ＰＩ标记,ＦＣＭ 检测细胞周期及细胞凋亡;双抗体夹心间接ＥＬＩＳＡ 法测定可溶性白介素２ 受体;Ｆｕｒａ２ 负载,双波长荧光测定法检测细胞内［Ｃａ２＋ ］ｉ;传代培养细胞测定细胞倍增时间。结果　低剂量辐射预照射可明显减轻其后大剂量照射对诸多淋巴细胞功能的抑制作用。结论　低剂量Ｘ 射线可诱导多参数免疫适应性反应。     

EGFR和NRF2对电离辐射导致的DNA损伤修复的影响

目的 探讨表皮生长因子受体（EGFR）和核转录因子E2相关因子2（NRF2）对人宫颈癌HeLa细胞受到电离辐射损伤后的DNA损伤响应及修复作用。 方法 将人宫颈癌HeLa细胞按2种处理方式分组：（1）采用小干扰RNA敲降HeLa细胞中的EGFR,采用137Cs γ射线照射源照射细胞。将HeLa细胞分为对照组（HeLa siCtrl）、敲降EGFR组（HeLa siEGFR）、照射组（HeLa siCtrl+8 Gy）、敲降EGFR+照射组（HeLa siEGFR+8 Gy）。采用免疫荧光实验（8 Gy照射后6、12、24 h）检测细胞中磷酸化组蛋白H2A变异体（γ-H2AX）foci的数量;采用实时荧光定量聚合酶链式反应（RT-qPCR）检测NRF2下游靶基因;采用流式细胞术检测EGFR对HeLa细胞周期的影响;采用核质分离实验分离HeLa细胞的胞质蛋白和胞核蛋白;采用蛋白质免疫印迹法检测NRF2、EGFR、血红素氧合酶1（HO-1）、共济失调毛细血管扩张突变基因Rad3相关激酶（ATR）Thr1989位点的磷酸化水平、检查点激酶1（CHK1）在Ser345位点的磷酸化水平。（2）采用小干扰RNA敲降HeLa细胞中的NRF2,采用137Cs γ射线照射源照射细胞。将HeLa 细胞分为对照组（HeLa siCtrl）、敲降NRF2组（HeLa siNRF2）、照射组（HeLa siCtrl+8 Gy）、敲降NRF2+照射组（HeLa siNRF2+8 Gy）。采用免疫荧光实验检测细胞中γ-H2AX foci的数量。符合正态分布的计量资料的组间比较采用两独立样本t检验（方差齐）。 结果 （1）8 Gy照射6、12、24 h后,HeLa siEGFR+8 Gy组细胞中γ-H2AX foci的数量均多于HeLa siCtrl组[（94.00±1.00）% 对（89.67±2.03）%、（72.33±1.76）% 对（60.00±1.73）%、（43.00±2.31）% 对（26.33±1.20）%],且差异有统计学意义（t=3.919、4.919、6.402,均P<0.05）。与HeLa siCtrl组比较,HeLa siEGFR+8 Gy组的细胞G2/M期阻滞显著受损[（46.53±3.06）%对（37.90±4.61）%],且差异有统计学意义（t=4.384,P<0.05）。与HeLa siCtrl组比较,HeLa siEGFR+8 Gy组的HO-1表达下降66.66%（1.35±0.10对0.45±0.02）,且差异有统计学意义（t=8.782,P<0.05）。敲降EGFR后细胞核内的NRF2蛋白水平降低,辐射引起的NRF2下游ATR-CHK1信号通路活化水平及HO-1蛋白水平均降低。（2）8 Gy照射6、12、24 h后,与HeLa siCtrl组相比,HeLa siNRF2+8 Gy组细胞中γ-H2AX foci的数量均多于HeLa siCtrl组[（96.67±0.88）%对（89.67±2.03）%、（77.33±1.20）% 对（60.00±1.73）%、（54.33±2.19）% 对（26.33±1.20）%],且差异均有统计学意义（t=3.166、4.919、11.220,均P<0.05）。 结论 电离辐射条件下,敲降EGFR可以减少NRF2蛋白入核,抑制ATR-CHK1信号通路激活及下游基因HO-1的表达,降低人宫颈癌HeLa细胞的DNA损伤修复能力。     

In vitro studies of cellular response to DNA damage induced by boron neutron capture therapy

The aim of these studies was to evaluate the mechanisms of cellular response to DNA damage induced by BNCT. Thyroid carcinoma cells were incubated with ¹⁰BPA or ¹⁰BOPP and irradiated with thermal neutrons. The surviving fraction, the cell cycle distribution and the expression of p53 and Ku70 were analyzed. Different cellular responses were observed for each irradiated group. The decrease of Ku70 in the neutrons +BOPP group could play a role in the increase of sensitization to radiation.     

Role for non-homologous end-joining in the repair of UVA-induced DNA damage

PURPOSE: The biological significance of long-wavelength ultraviolet (UV) light, UVA, is increasingly realized, but the precise nature of the cellular damage responsible for the effects of this radiation is still not clear. It has been reported that UVA can induce double-strand breaks in DNA, but the biological significance of these is not known. We have therefore examined the UVA sensitivity of a cell line deficient in non-homologous end-joining, the major pathway for the repair of DNA double-strand breaks in mammalian cells in order to determine the biological importance of UVA-induced DSB. MATERIALS AND METHODS: Xrs-6, a Chinese hamster ovary cell line mutant for XRCC5 (Ku80) was compared with its parental CHO-K1 cell line for its sensitivity to UVA radiation (365 nm) using both a clonogenic assay and the micronucleus assay. RESULTS: Xrs-6 cells were sensitive to the cytotoxic effects of UVA. This resulted in the formation of chromosome damage, as measured by the micronucleus assay, which this cell line was unable to repair. CONCLUSIONS: Owing to the nature of the repair defect in these cells, these results imply that DNA double-strand breaks are produced in cells following UVA irradiation, that the non-homologous end-joining repair pathway is involved in their repair and that they are produced with sufficient frequency to have biological significance.     

Role for non-homologous end-joining in the repair of UVA-induced DNA damage

Purpose : The biological significance of long-wavelength ultraviolet (UV) light, UVA, is increasingly realized, but the precise nature of the cellular damage responsible for the effects of this radiation is still not clear. It has been reported that UVA can induce double-strand breaks in DNA, but the biological significance of these is not known. We have therefore examined the UVA sensitivity of a cell line deficient in non-homologous end-joining, the major pathway for the repair of DNA double-strand breaks in mammalian cells in order to determine the biological importance of UVA-induced DSB. Materials and methods : Xrs-6, a Chinese hamster ovary cell line mutant for XRCC5 (Ku80) was compared with its parental CHO-K1 cell line for its sensitivity to UVA radiation (365 nm) using both a clonogenic assay and the micronucleus assay. Results : Xrs-6 cells were sensitive to the cytotoxic effects of UVA. This resulted in the formation of chromosome damage, as measured by the micronucleus assay, which this cell line was unable to repair. Conclusions : Owing to the nature of the repair defect in these cells, these results imply that DNA double-strand breaks are produced in cells following UVA irradiation, that the non-homologous end-joining repair pathway is involved in their repair and that they are produced with sufficient frequency to have biological significance.