【作者】 张青；

【导师】 周淑芸；

【作者基本信息】 第一军医大学 ， 内科学血液专业， 2001， 博士

【摘要】 对肿瘤发病机制的研究一直是医学领域研究的热点之一，但以往对发病机制及其演进过程的研究多集中于肿瘤相关基因的检测，而对于遗传物质在细胞核内的空间构象的变化与肿瘤的发生及发展是否还存在着相关性却未予足够重视。70年代末就有人提出了每个染色体在核内都占有一个特定的空间区域，称为染色体结构域。为此人们还提出了许多的数学模型，来解释染色体的拓扑结构同其功能的关系。多数人认为，染色体结构域在核内位置相对恒定，但在细胞周期循环中也呈现有规律的变化。在肿瘤发生时，其本身的拓朴结构，包括体积，表面积、形状以及在核内的空间位置都会发生一系列的变化。而肿瘤中常出现的染色体易位也与染色体在核内构象有关，相邻染色体间发生易位的频率要大于非相邻的染色的易位，也就是说染色质在核内的空间定位决定了易位及肿瘤的发生概率。 慢性髓细胞性白血病（CML）是起源于单一克隆的造血系统恶性肿瘤疾病，在95％的CML患者都可检测到特异的ph染色体，它是第9、22号染色体相互易位而成，即9号染色体长臂上的c-abl与22号染色体长臂的断裂点集中区（BCR）相互易位形成bcr-abl融合基因，其编码的融合蛋白具有酪氨酸激酶活性，是CML发病机制中的主要因素。CML在临床上分为慢性期、加速期和急变期，它们在细胞遗传学及分子生物学上存在着较大异质性，在急变期常会出现额外的染色体和基因的变异。但是这些异常是否与相关染色体在核内的拓朴结构的改变有关尚不清楚，这些变化在CML的发生和演进中的意义如何，目前也无确凿的证据。 我们利用荧光原位杂交和激光共聚焦扫描显微镜方法，选用bcr，abl，c－mmc和的3单拷贝恻A探针，第 8，9，17和 22号全染色体探针，观察了与慢粒相关的遇传物质在间期核内拓扑学的表现和变异情况。结果发现：在人淋巴细胞株IM－9和KapZ细胞中，相关基因在问期核内的三维空间定位呈非随机分布，her和abl基因之问的融合同二者在核内的分布区域的一致性是有关的，提示基因之间的融合频率同其在核内三维空间相互位置呈正相关；同源染色质的拓扑学表现具有差异性，井且在细胞周期循环中表现出动态的变化。染色质的凝集和松散结构同染色质的活性密切相关，同源染色质之间的凝集程度存在着差异性。放射可以促使 IM－9细胞中比 和 abl基因相互接近，同时能改变第 9，22号染色质的形状和凝集程度，促使二者更加松散和伸展，活性增加，并使居于其上的abl和伙 基因的空间位置发生改变，使碍二者之间更加接近，这可能是放射引起her和abl融合的原因之一。 CML是一种恶性克隆增殖性疾病，几乎每个病例最终都会发生急性变。在慢粒急变时，C－CyC和 P53基因在细胞核内的空间定位会发生明显的变化，都有一个向心性的移动过程，同时，第8和17号染色质的拓扑结构也发生了明显的变化，其表现更接近于K562细胞。这些结果提示，旷myc和的3在细胞核内空间定位的变化以及第8和17号染色质拓扑形态的改变同慢粒的急变之间存在着明显的相关性，这些基因或染色体的拓扑学的变异情况有可能成为判断慢粒急变的检测指标之一。更多还原

【Abstract】 The Study of Topology of genetic material relative tochronic myeloid leukemia in interphase nucleousThe study to neoplasm morbility mechanism is heat subject at all time in medical territory. But most of the studies have been focused on variations of the genes relative to tumor, meanwhile, the relativity between topologic changes of genetic material in the nuclear spaces and the tumor genesis and evolution has not been pay much attention. In the end of 70’s, a presumption has been raised that each chromatin occupies a territory in the nucleus, which being called chromatin territory. Many mathematics model have been submitted to explain the relationship between chromatin topologic structure and its function. Most people believe that chromatin territory locations in nucleus are constant, and exhibit regular changes in cell cycle. In the genesis of tumor, a series of variations of the chromatin territory topology, including volume, surface area and the location in nuleus space, may take place. The familiar chromosome translocations in tumor are correlate to their conformation in nucleus. Adjacent chromosomes translocation are more frequent than non-adjacent chromosomes, that means chromosome locations in the space of nucleus determine the frequency of translocation and tumor genesis.Chronic myeloid leukemia (CML) is a hematopoietic therioma deriving from a singleness clone, ph chromosome, existing in 95% CML, is formed by reciprocal translocation of chromosome 9 and 22. c-abl gene in the long arm of chromosome 9 and break point concentration region (BCR) in the long arm of chromosome 22 translocate and form bcr-abl fusion gene, which code p210fusion protein having tyrosine kinase activity and; is a key in CML genesis. In clinical, CML has been divided into chronic period, acceleration period and blast period. Each period has distinct heterogenicity, and, .extra variations of chromosomes and genes emerge in bla$t period. But we still do not know if these variations are related to the changes of chuomatin topology in nucleus, and what kind of rules of these variations in CMLigenesis and evolution.Conbined with flurescence in situ hybridization (FISH) and confoeal laser scanning microscope (CLSM), we observed the topologic characters of genetic material relative to CML using single copy sequence bcr, c-abl, c-myc and p53 DNA probes, chromosome 8, 9, 17 and 22 whole chromosome probe(WCP). The results show that all genes are distributed non-randomly in 3-dimension space of interphase nucleus of IM-9, human lymphocyte cell line, and K562 cell line. The fusion of bcr and abl genes are consistent with their distribution in the same ugion which prompts that the fusion frequency of genes are positive correlate to their relative 3-dimension location’ in the interphasenucleus. Homogenous chromatin exhibit diversities in topologic characters and dynamic changes in cell cycle. Chromatin condensation and decondensation have a intimate relationship with its activity, and homologous chromatin exist differences in chromatin condensation degree. Irradiation may facilitate approximability of bcr and abl genes in the nucleus of IM-9 cell, alter the figure and condensation of chromosome 9 and chromosome 22, making them more extent and active. These results could explain irradiation can lead to the fusion of bci and abl genes.CML is a kind of malignancy clone proliferation disease. Almost all cases will lead to blast. When the occurrence of blast, c-myc and p53 genes location in the interphase of nucleus may change obviously. They movetoward to the nuclear center at blast period. Meanwhile, the topology of chromosome 8 and chromosome 17 vary dramatically and be more approximating to K562 cell. These results hint that the location variation of c-myc and p53 genes in nuclear space and the topologic changes of chromosome 8 and chromosome 17 are relative to CML blast. The variation of these genes and chomatin could be indicator to estimate CML blast.更多还原