【作者】 周平坤；

【导师】 吴德昌；

【作者基本信息】 中国人民解放军军事医学科学院 ， 卫生毒理学， 2004， 博士

【摘要】 致癌是辐射主要的生物效应之一，释放α粒子的放射性核素已被广泛应用，而且，在人们居住和日常生活或工作环境也可能存在释放α粒子氡及其子体。由此，α粒子致癌问题，已越来越受到人们重视，开展这方面的机理和应用基础研究，具有非常重要的现实意义。 本研究围绕DNA修复和细胞周期检测点调控两大主要的细胞学反应机制，开展了α粒子辐射致癌的遗传不稳定性机理研究，获得如下主要结果： (1) 我们在本实验室完成的α粒子诱发人支气管上皮细胞BEP2D恶性转化模型的工作基础上，对在裸鼠体内成瘤的癌变细胞群体在体外培养进行克隆化，建立了α粒子诱发人支气管上皮细胞恶性转化细胞的5个亚克隆系，癌变细胞克隆无论是细胞形态还是增殖生长速度等都发生了明显的变化，为深入探讨癌变机理提供了更理想的实验模型。 (2) 通过分析比较各癌变克隆细胞系与亲本BEP2D细胞核型特征，发现各癌变克隆细胞系均发生1条13号染色体和Y染色体丢失、1条2号染色体和12号染色体的长臂增加，不同癌变克隆系还有各自的特征。发现染色体数目不稳定性是辐射诱发癌变细胞的最显著的遗传学变化特征之一，多倍体核型发生率是呈进行性发展，最明显的是恶性转化细胞BERP35T4，克隆后第5代多倍体率达40.5％，第32代上升到约65％。 (3) 分析比较了癌变细胞系BERP35T1和BERP35T4对γ射线的敏感性变化，结果表明α粒子诱发癌变细胞的电离辐射敏感性增加，DNA链断裂修复能力降低。通过cDNA微矩阵、PCR-单链构像多态性和Northern杂交技术检测发现，辐射细胞在恶性转化早期(第25代以前)，DNA修复基因XRCC-5、DNA-PKcs的表达就受到抑制，XRCC-5基因发生了碱基突变；恶性转化细胞中修复基因XRCC-2，XRCC-3、XRCC-5、ERCC-2，ERCC4、EX01，APEX、UNG、NBS1、NTHL1等表达受到明显抑制，这些基因表达产物涉及到DNA链断裂修复、碱基和核苷酸切除修复途径。由此提出，α粒子启动细胞恶性转化的机理之一，就是首先改变了维持细胞遗传稳定性的DNA修复机制，使照射细胞成为“活跃突变”表型。军事医学科学院博士论文 但也有部分基因特别是DNA一PKcs在细胞恶性转化之后表达明显增加，而且DNA一PKCS表达增加在人肺癌组织和肝胆管肿瘤组织中也得到证实。 (4)研究发现，多倍体发生率高、染色体极不稳定的癌变细胞系BERP35T4，在纺锤体微管蛋白受到噬氨酷哒哇破坏后，不能正常启动纺锤体检测点机制，即细胞不能有效地终止有丝分裂进程，而且该细胞中有丝分裂检测点相关基因以斤的启动子区CpG岛发生了甲基化，并导致了该基因的表达沉默。表明该细胞的纺锤体检测点机制发生异常，这将是导致该细胞系染色体不稳定性的重要原因之 (5)为了观察纺锤体结构受损后的细胞凋亡反应，我们采用三种荧光染料复染细胞，即Hoechst 33258染活细胞和凋亡细胞核，PI染坏死细胞核，FDA染活细胞和凋亡早期细胞胞浆，并利用松胞素B阻止细胞分裂、形成双核，以识别细胞凋亡与有丝分裂时相的关系。结果发现，细胞纺锤体微管蛋白受到一定剂量唆氨酷哒哇破坏时，癌变细胞BERP35TI和BERP35T4的凋亡发生率要显著低于正常细胞。而且正常细胞的凋亡主要发生在细胞有丝分裂前，癌变细胞特别是BERP35T4细胞凋亡是发生在有丝分裂之后，双核凋亡细胞比例明显增加，也就是发生在第二代细胞以后。由此表明，部分恶性转化细胞可能起源于凋亡机制异常的细胞克隆，由此致使子代细胞出现亚二倍体和多倍体的遗传不稳定性表型，促进细胞恶性转化进程。更多还原

【Abstract】 Carcinogenesis is one of the major biological effects induced by ionizing radiation. a particle emitting isotopes are now widely used in the nuclear industries, could even be used by the terrorisms to construct the DDR or dirty bomb. A number of epidemiological studies indicate that radon is an important environmental factor that is associated with the development of human bronchogenic carcinoma. Although the close association between radon exposure and lung cancer has been revealed, the detailed mechanisms of radon-induced lung cancer are still unclear.Cell cycle checkpoints and DNA repair are thought to be essential for ensuring the genome or chromosomes integrity. Current study was to investigate the changes of cell cycle checkpoints, DNA repair and disrupted spindle-induced apoptotic responsiveness in the malignant transformed cell lines of human bronchial epithelial BEP2D cells generated by a particle exposure. The results were summarized below:1) Tumor cells were isolated from nude mice generated by implanting passage 35 cells of a -particles-irradiated BEP2D cells and were subcloned in vitro. Five individual malignant transformed cell lines named as BERP35T-1 / -2 / -4 / -5 / -6 were subcloned. The malignant transformed phenotype was further manifested by cellular morphological changes, increased cell growth, cytogenetic changes, etc.2) The cytogenetic characteristics of five individual malignant transformed cell lines were analyzed. Deletions of one copy of chr13 and chrY, and an unidentified fragment attached on the long arms of chr2 and chr12 respectively were observed in all the malignant transformed cell lines. The ratio of polyploids of BERP35T-4 cells was about 40.0 % at passage 5 and enhanced to 65%, significant higher than that of PEB2D cells.3) Transformed cell lines BERP35T1 and BERP35T4 showed a markedly lower capacity of v -ray-induced DNA DSBs rejoining and highly increasedradiosensitivity than parental BEP2D cells. The analysis of gene express (mRNA) level by DNA microarray, RT-PCR and Northern blot hybridization revealed a significant depressed expression of the DNA repair genes XRCC-2?XRCC-3? XRCC-5?ERCC-2?ERCC4?EXO1?APEX?UNG?NBS1?NTHL1 in the malignant transformed cells. However, an increased expression was detected in transformed BERP35T4 cells as well as in human non small lung cancer tissues and in hepato- and cholangio- neoplasm.4) The mitotic spindle checkpoint and apoptosis in response to nocodazole, a microtubule-disrupting agent, were investigated in a particles-transformed human bronchial epithelial cell lines BERP35T1, BERP35T4 and the parental BEP2D cells. When treated with 0.2ug/ml of nocodazole, BEP2D and BERP35T1 cells were efficiently arrested in mitotic phase, whilst BERP35T4, a transformed cell line showing chromosomal instability, failed to arrest, and demonstrated a low G2/M fraction and a high aneuploidy ratio, indicating that BERP35T4 cells has an defect in spindle checkpoint function. Methylation of 5’CpG island of Chfr gene, a mitotic checkpoint gene that functions in early prophase to delay chromosome condensation, was found in BERP35T4 cell but not in BEP2D cells. As coincident with methylation status, the expression of Chfr gene was markedly suppressed.5) The extent of apoptosis induced by nocodazole (0.3ug/ml) was significantly higher (2~2.5 fold) in BEP2D cells than that in two transformed cell lines. Furthermore, the apoptosis was found to take place dominantly before mitotic division in BEP2D cells but half of them after mitotic division in BERP35T4 cells, the later was characteristic of increased ratio of dinucleated apoptotic cells when co-treated with cytochalsin B.更多还原