癌组织中的遗传不稳定性的RAPD分析

目的　检测肿瘤发生发展过程中DNA和染色体的不稳定性及筛选与新的癌基因或抑癌基因相关的分子标志。方法　用 9个随机引物对 5种肿瘤共 12 8个样本进行RAPD分析 ,检测其DNA和染色体的不稳定性。不稳定性的扩增带被切割回收纯化 ,克隆 ,然后被标记为探针作Southern和Northern印迹杂交分析 ,再进一步测序分析。结果　在所有的癌组织标本中胃癌样本 5号和 3号显示出最高的遗传变异。这 5种癌组织中遗传不稳定性的平均检出率分别从 4 2 2 %到 4 9 4 %。每个随机引物的检测能力有极显著差异 ,分别从 2 7%到 6 8%不等。其中引物2最高达 6 8% ,引物 8最低 ,为 2 7%。图 2中的B带为一个单拷贝片段经RFLP分析后发现其在胃癌和结肠癌中是缺失突变 ,测序后 ,经查询是一个新的序列并被GeneBank收录 (登录号AF15 10 0 5 )。进一步研究显示它可能是一个新抑癌基因的调控元件部分 ,含有一个顺式作用的“CACA”启动子 ,一个“GATA”家族序列的增强子和一个转录起始密码子。结论　RAPD与其他方法结合使用能检测肿瘤发生过程中DNA和染色体的不稳定性 ,并筛选与新的癌基因或抑癌基因相关的分子标志。

Genetic instability in cancer tissues analyzed by random amplified polymorphic DNA PCR

Objective To detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. Methods Five kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned, labeled as probes for Southern and Northern blot analysis and DNA sequencing. Results Sample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40% (42.2%-49.4%). There were significant differences in the ability of each primer to detect genomic instability ,which ranged from 27% (primer 8) to 68% (primer 2). Band B is a single copy fragment ,and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005. Further analysis revealed that it might be part of a cis- regulatory element of a new tumor suppressor gene, containing a promoter of cis-action "CACA" box, an enhancer of "GATA" family and a start codon. Conclusions It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.