【作者】 左雅慧；

【导师】 童建； 王仲文；

【作者基本信息】 苏州大学 ， 卫生毒理学， 2010， 博士

【摘要】 目的:在细胞、基因和蛋白水平上检测60Co-γ射线诱发人肝细胞基因组的不稳定性,并用基因芯片和双向电泳-质谱技术探讨其分子机制,筛选与辐射诱发基因组不稳定性相关的基因和蛋白,为阐明辐射诱发基因组不稳定性的分子机制提供基础资料。方法:(1)采用60Co-γ射线照射人正常肝细胞7702(HL-7702),照射剂量为0 Gy(对照组)、2 Gy、4 Gy、6 Gy、8 Gy、10 Gy。检测受照后7702细胞克隆存活率、微核形成率、单细胞凝胶电泳(SCGE)、细胞凋亡率。(2)检测各剂量点受照细胞子代的上述指标。(3)对各剂量受照细胞克隆子代同时给予2Gy的二次照射,然后检测克隆存活率、微核形成率、单细胞凝胶电泳、细胞凋亡率。(4)分别提取经2、4、6 Gyγ射线照射后子代细胞的总RNA,采用Illumina人全基因组基因芯片分析基因表达情况,并筛选出差异表达基因。(5)用实时荧光定量PCR(RT-PCR)技术验证部分差异基因。(6)用GeneSpring GX 10软件对差异表达基因进行生物信息学分析,并构建差异表达基因相互作用网络。(7)提取受照射后子代细胞的总蛋白进行2-DE分离,考马斯亮蓝染色,差异表达点进行MALDI-TOF质谱分析,NCBInr数据库搜索鉴定分析结果。(8)用Western blot方法验证质谱鉴定出的差异表达蛋白热休克蛋白60(HSP60)和珠蛋白转录因子1(GATA-1)在各剂量克隆子代中的表达。(9)用激光共聚焦显微技术观察差异表达蛋白HSP60、GATA-1和真核翻译起始因子5A (EIF5A)在各剂量克隆子代中的定位及表达。结果:(1)首次照射后,HL-7702细胞的克隆存活率、微核形成率、SCGE尾长、细胞凋亡率与照射剂量之间存在明显的剂量效应关系。(2)首次照射后各剂量组存活的克隆子代细胞经传代培养后,克隆存活率、微核形成率、SCGE尾长与对照组无显著差异。(3)首次照射后各剂量组存活的克隆子代细胞经2Gy的二次照射后,上述检测结果与首次照射剂量之间存在剂量效应关系。(4)基因芯片测定2Gy照射后子代细胞差异表达显著的基因有262个;4Gy照射组有2746个差异表达基因;6Gy照射组有3406个差异表达基因;三个剂量组的共同差异表达基因有71个,其中上调基因35个,下调基因36个。这些基因的功能涉及细胞周期、细胞骨架和运动、细胞凋亡、DNA结合、细胞信号转导、代谢、DNA复制和修复等。利用生物信息学分析软件构建了差异表达基因相互作用网络图并分析了RAN、CDT1、IER3、V-FOS等基因的生物学功能。(5)受照射7702细胞克隆子代细胞双向电泳图谱与对照组相比,共发现差异蛋白点42个,其中10个上调蛋白,32个下调蛋白。经质谱分析,成功鉴定出17个差异表达蛋白,这些差异蛋白包括翻译控制肿瘤蛋白(TCTP)、热休克蛋白27(HSP27)、热休克蛋白60(HSP60)、珠蛋白转录因子1(GATA-1)、巯基特异性抗氧化酶(TSA)、氯离子通道蛋白1(CLIC1)、真核翻译起始因子(EIF1A, EIF5A)、真核翻译延长因子1(EEF1A)等。(6)Western blot分析结果表明HSP60和GATA-1表达与受照剂量的关系与2-DE结果一致。(7)激光共聚焦结果显示HSP60与EIF5A蛋白在细胞中表达丰富,主要分布于细胞质中细胞核的周围。荧光定量分析结果与双向电泳分析结果一致。结论:(1)电离辐射诱发的基因组不稳定性可传递给受照细胞的后代,并在细胞复制多代后仍以潜在的方式存在于子代中,从而表现出滞后的遗传学效应。基因组不稳定性的发生与DNA的首次损伤事件之间存在明显的相关关系。(2)二次事件的放大作用在基因组不稳定性的传递过程中起着重要的作用。二次损伤放大了处于不稳定状态的基因组损伤,使其更容易被检测,因而可作为研究基因组不稳定性的有效工具。(3)电离辐射可诱发HL-7702子代细胞中一系列基因与蛋白质表达的改变,提示基因组不稳定性涉及复杂的调控机制。其中,RAN、CDT1、IER3、RAD51AP1、HAVCR2基因及HSP60、GATA-1蛋白在受照肝细胞子代中均显示出特征性的差异表达,有望成为辐射诱发基因组不稳定性的分子生物学标志。更多还原

【Abstract】 Objective: Radiation-induced genomic instability(RIGI) in human liver cells was detected at the cellular, molecular and proteomic level. cDNA chip and proteomic analysis was conducted upon progeny of irradiated human liver cells to provide experimental data for exploring the molecular mechanism of RIGI.Merhods: (1) The cloning efficiency, micronucleus frequency and apoptosis efficiency of human liver cells 7702(HL-7702) irradiated by 60Co-γrays were detected, and the method of single cell gel electrophoresis(SCGE) was used to measure DNA chains damage. (2) The progeny of HL-7702 cells were irradiated by 0, 2, 4, 6, 8, and 10Gy of 60Coγ-irradiation, and the effects mentioned above were detected in the progeny of the irradiated cells. (3) The progeny were secondly irradiated with 2Gy of 60Coγ- irradiation, and the delayed effects were detected. (4) cDNA gene chip was used to measure the transcriptional profile in progeny of HL-7702 cells exposed to 0, 2, 4, and 6Gy 60Coγ-irradiation, and the differentially expressed genes were further identified by Quantitative real-time PCR. A pathway-based network was constructed using a software (Genespring GX10) to analyze the functional relations among the differential genes. (5) Two-dimensional electrophoresis(2-DE) was used to screen the proteins differentially expressed in the progeny of human liver cells surviving from ionizing radiation, and mass spectrometry was used to identify the protein-spots significantly altered in expression. (6) The differential expression proteins of GATA-1, HSP60 were verified by Western blot. (7) Laser confocal scanning microscopy(LGSM) was used to detect the differential expression proteins of EIF5Aand HSP60 to confirm the 2-DE result.Results: (1) The cloning efficiency decreased with the increase of doses after the initial irradiation, while micronucleus frequency, percentage of apoptosis and comet rate increased with the increase of doses. (2) Damage of the survival cells secondly irradiated was correlated with the original irradiation doses. (3) A total of 71 differentially expressed genes were screened, most of which associated with transduction, cell cycle regulation, cellular immunity, cytoskeleton and movement, cell replication and repair mechanism. A pathway-based network was constructed to reveal the biological functions of RAN、CDT1、IER3、V-FOS.(4) A total of 42 differentially expressed proteins from the progeny of irradiated cells were screened, of which 17 were identified by MALDI-TOF-MS analysis, including 4 up-rugulated and 13 down-regulated proteins. The up-regulated expression of two proteins, mitochondrial heat shock 60kD protein (HSP60) and globin transcription factor 1 (GATA-1), were further confirmed by immunoblotting. (5) The differentially expressed proteins of HSP60 and EIF5A were localized in the cytoplasm, and the expression of HSP60 was up-regulated in the progeny of irradiated cells in a dose-dependent manner, which was consistent with the result of 2-DE.Conclusions: (1) Radiation-induced genomic instability may sustain in the progeny of surviving cells. The delayed damage after a second irradiation was correlated to the original irradiation dose. (2) A second irradiation plays an important role in transforming the genomic instability to the progeny, to make the damage more easily to be detected. (3) Irradiation can induce differentially expressed genes and proteins in the progeny of irradiated cells, which may be applied as potential biomarkers of radiation damage in future studies.更多还原