双微体的分子细胞遗传学研究

发布时间：2018-01-13 23:18

本文关键词：双微体的分子细胞遗传学研究　出处：《吉林大学》2012年博士论文　论文类型：学位论文

【摘要】：双微体是游离于染色体外的小片段球状染色小体，具有独特的自我复制功能，见于乳腺癌、肺癌、卵巢癌、结肠癌，尤其是神经母细胞瘤等多种人类肿瘤细胞，是基因扩增的一种表现形式，常携带耐药基因或原癌基因，导致肿瘤相关蛋白的过表达，针对携带基因的靶向治疗对肿瘤的预后和治疗具有重要的意义。尽管有关双微体的研究很多，但专门针对各种肿瘤的双微体染色体来源的研究很少，我们从ATCC公司筛选所有携带双微体的肿瘤细胞系，进行细胞培养，并在第3代、8代、13代时获取细胞。利用染色体核型分析技术检测双微体的数目及分布情况及粗略的细胞遗传学特征，发现不同肿瘤细胞系所携带双微体的数目及分布相差极大。继而通过arrayCGH获得扩增的基因，并明确其断裂点，采用相应的DNA探针，利用荧光原位杂交确认基因扩增并进一步确认染色体的来源及明确扩增的形式。我们发现在10个细胞系中，血液肿瘤占10%（HL-60细胞系），MC-IXC细胞系，COLO-320DM细胞系，SW480细胞系及HL-60细胞系中由于MYC扩增基因所致，双微体广泛存在，但其扩增区域的断裂点不同，其中前2个细胞系MYC双微体与MYC均质染色区共存，后2个细胞系仅存在MYC双微体。SW1783细胞系、5637细胞系、CAMA-1细胞系的扩增区域分别包含PDGFRA基因、RAF1基因及SOX4基因、MYEOV基因及CCND1基因，均以均质染色区的形式存在。T84细胞系、SNU-1细胞系及NCI-N87细胞系仅在部分细胞中见少量双微体，arrayCGH未见明显扩增的区域，未行扩增基因FISH检测。得出主要以下结论： 1.同一细胞内的双微体及均质染色区可携带不同来源的基因，病理类型相同的肿瘤细胞可携带不同来源的双微体。 2.携带相同原癌基因的不同肿瘤，双微体的断裂点不同； 3.双微体拥有除均质染色区以外的形成机制； 4.不同代细胞间的基因扩增未见明显改变； 5.来源于不同染色体的基因可以共同交替扩增，形成一个新的混合的扩增区域。并验证了以下结论 1.双微体多见于实体肿瘤细胞系中，血液肿瘤中罕见； 2.携带双微体的细胞多伴有复杂的染色体核型改变； 3.不同的肿瘤细胞系携带来源不同的双微体，数目相差巨大； 4.在某些肿瘤细胞系中，，双微体与均质染色区可以相互转化。主要创新点：（1）首次筛选不同的包含双微体的实体肿瘤细胞系及血液肿瘤细胞系共同比较研究。（2）联合应用染色体核型分析、arrayCGH及FISH技术，准确有效的找出扩增基因及扩增形式。（3）利用arrayCGH技术明确扩增区域的断裂点。
[Abstract]:Double microbodies are small globular chromatid bodies which are free from chromosomes. They have unique self-replicating function and can be seen in many kinds of human tumor cells such as breast cancer lung cancer ovarian cancer colon cancer especially neuroblastoma and so on. It is a form of gene amplification, often carrying drug resistance genes or proto-oncogenes, resulting in over-expression of tumor-associated proteins. Targeted therapy for carrying genes is of great significance to the prognosis and treatment of cancer. Although there are many studies on double microsomes, there are few studies on the origin of double microsomes specifically for various kinds of tumors. We screened all tumor cell lines with double microsomes from ATCC and cultured them for 8 passages in the third generation. Chromosome karyotype analysis was used to detect the number and distribution of double microbodies and the rough cytogenetic characteristics. It was found that the number and distribution of double microbodies carried by different tumor cell lines varied greatly. Then the amplified genes were obtained by arrayCGH and the breakpoints were identified and the corresponding DNA probes were used. Fluorescence in situ hybridization (Fish) was used to confirm the origin of chromosome and the form of chromosome amplification. We found that blood tumor accounted for 10 HL-60 cell lines out of 10 cell lines. The MC-IXC cell line, COLO-320DM cell line, SW480 cell line and HL-60 cell line, due to the MYC amplification gene, the presence of double microbodies is widespread. However, the breakpoints of the amplified region were different. The first two cell lines, MYC double microbodies and MYC homogenized staining region, only existed MYC double microbody. SW1783 cell lines. The amplified region of 5637 cell line, CAMA-1, contains PDGFRA gene, RAF1 gene, SOX4 gene, MYEOV gene and CCND1 gene, respectively. There were only a few double microbodies in some cells of SNU-1 cell line and NCI-N87 cell line in the form of homogeneous staining region. There was no obvious amplified region in arrayCGH, and no amplification gene FISH was detected. The main conclusions are as follows: 1. Double microbodies and homogenous staining regions in the same cell can carry genes from different sources, while tumor cells with the same pathological type can carry double microbodies from different sources. 2. Different tumors carrying the same proto-oncogene had different breakpoints. 3. The double microbodies have the formation mechanism except the homogeneous staining area. 4. There was no obvious change in gene amplification between different generations. 5. Genes from different chromosomes can be amplified alternately to form a new mixed region. And verify the following conclusions 1. Double microbodies were found mostly in solid tumor cell lines, but rare in blood tumors. 2. The cells carrying double microbodies were often accompanied by complex chromosome karyotype changes; 3. Different tumor cell lines carry different double microbodies, and the number of tumor cells varies greatly. 4. In some tumor cell lines, the double microsomes and the homogenized staining region can transform each other. Major innovations: For the first time, different solid tumor cell lines containing double microbodies and blood tumor cell lines were screened for the first time. (2) using chromosome karyotype analysis and FISH technique, we can find out the amplified genes and their forms accurately and effectively. ArrayCGH technique was used to identify the breakpoints of the amplified region.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R394

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