【作者】 王会平；

【导师】 周平坤；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 目的：研究小于0.5Gy低剂量辐射对基因表达的影响规律，筛选并鉴定低剂量辐射反应基因，为发展低剂量辐射暴露的分子标志物奠定基础；探讨其中CHD6基因表达变化对细胞增殖、辐射敏感性的影响及其机理。 方法：应用基因芯片技术对0.05、0.2和0.5Gy ⁶⁰Coγ射线照射正常人淋巴母细胞AHH-1的全基因组mRNA表达水平进行对比分析，RT-PCR、real-time PCR和Northern blot验证部分差异表达基因mRNA的表达水平；利用质粒介导的siRNA技术，建立CHD6基因表达抑制细胞模型，RT-PCR检测CHD6 mRNA的表达，细胞生长曲线和流式细胞技术分别检测细胞增殖及细胞周期的变化，荧光染色法检测细胞凋亡，细胞克隆形成率检测细胞辐射敏感性，GST-pull down检测CHD6相互结合蛋白。 结果：1)在所分析的14，112个基因中发现，0.05Gy照射组差异表达2倍以上基因有43个，其中表达上调的有25个，表达下降的有18个；0.2Gy照射组差异表达显著的基因有83个，其中表达上调的有21个，表达下降的62个；0.5Gy照射组差异表达显著的基因有75个，表达上调的基因30个，表达下降的基因45个。 2)RT-PCR对部分基因的辐射诱导表达进行了验证，包括0.05Gy照射组的BMPR2、CONNEXIN43、LYK5和NOL6基因表达上调，CCNB1IP1、SDPR、CCT5和KIAA0231基因表达下降；0.2Gy照射组的MAPK14、NDST1、CENPF和SELP基因表达上调，APOLLON、RNF2、VTN和PCDH18基因表达下降；0.5Gy照射组的STAT3、GPR56、MIZF、ATP9A、CAMKK2和CHD6基因表达上调，LCP1、XRCC4、RLF3基因表达下降。这些基因的表达变化与基因芯片的筛选结果一致。 3)利用real-time PCR对其中的XPC基因的辐射诱导表达的剂量效应规律进行了细致的分析，结果表明，其mRNA表达水平具有显著剂量依赖性，在至少0.05～10Gy范围内，随剂量的升高而表达丰度上调。而且XPC基因表达在0.05Gy照射后10h和2Gy照射后4h分别达到最高值，而紫外线和顺铂对XPC基因mRNA表达水平无明显影响。MSN、LCP1和AK123575基因尽管也受低剂量辐射诱导表达，但剂量效应关系不明显。 4)CHD6基因是本实验室以前通过差异显示PCR鉴定的一个0.5Gy辐射反应基因，更多还原

【Abstract】 Objective: To reveal the effects and regulation of low dose radiation on global gene transcription, and to screen and identify the differentially transcribed genes for developing novel molecular biomarkers or biodosimeter of ionizing radiation exposure; To investigate the roles of low dose responsive gene CHD6 on the proliferation and cellular radiosensitivity. Methods: cDNA microarray was used to detect the global transcriptional profiling of human lymphoblastoid AHH-1 cells at 4 h after exposure with ⁶⁰Co γ-ray at doses of 0.05, 0.2 and 0.5Gy. The expression changes of mRNA were confirmed by RT-PCR, real-time PCR and northern blot analysis. CHD6-silenced A549 cell model was generated by plasmid mediated siRNA technology. Growth curve, colony-forming ability, flow cytometry and fluorescent staining methods were used to measure the cell proliferation, radiosensitivity, cell cycle and apoptosis respetively. The interacting proteins of CHD6 were identified by GST-pull down and peptide mass fingerprint.Result: 1) Microarray containing the cDNA probes corresponding to 11,412 human genes was used. The results revealed that the transcription level of 43 genes were markedly alterated in 0.05Gy irradited cells, among which 25 genes were up-regulated and 18 genes were down-regulated. In 0.2Gy irradiated cells, 21 up-regulated genes and 62 down-regulated genes were screened. In 0.5Gy irradiated cells, a total of 75 differentially expressed genes were identified, there include 30 up-regulated genes and 45 down-regulated genes.2) The expression changes of a number of genes, as indicated in the microarray analyses, were further confirmed by semi-quantitative RT-PCR, including the upregulation of BMPR2, CONNEXIN43, LYK5, NOL6 genes, and downregulation of CCNB1IP1, SDPR, CCT5, KIAA0231 genes induced by 0.05Gy; upregulation of MAPK14, NDST1, CENPF, SELP genes, and downregulation of APOLLON, RNF2, VTN, PCDH18 genes induced by 0.2Gy; upregulation of STAB, GPR56, MIZF, ATP9A, CAMKK2, CHD6 genes, and downregulation of LCP1, XRCC4, RLF3 by 0.5 Gy irradiation.3) Using quantitative real-time PCR, we also revealed that the expression level of XPC更多还原