利用染色体区段混合BAC探针鉴定食管癌细胞中的染色体畸变

doi:10.3724/SP.J.1005.2014.0558

遗传 ›› 2014, Vol. 36 ›› Issue (6): 558-565.doi: 10.3724/SP.J.1005.2014.0558

利用染色体区段混合BAC探针鉴定食管癌细胞中的染色体畸变

郝佳洁¹, 王春丽^{1, 2}, 顾文跃^{1, 3}, 程潇钰¹, 张钰¹, 徐昕¹, 蔡岩¹, 王明荣¹

1. 中国医学科学院北京协和医学院肿瘤医院肿瘤研究所, 分子肿瘤学国家重点实验室, 北京 100021;
2. 安徽医科大学组织胚胎学教研室, 合肥 230032;
3. 盐城市第三人民医院病理科, 盐城 224001

收稿日期:2014-01-27 修回日期:2014-03-14 出版日期:2014-06-20 发布日期:2014-05-28
通讯作者: 王明荣,博士,研究员,研究方向：肿瘤遗传学。E-mail:wangmr2015@126.com E-mail:hjj8173@126.com
作者简介:郝佳洁,博士,助理研究员,研究方向：肿瘤遗传学。E-mail:hjj8173@126.com
基金资助:
国家自然科学基金项目(编号：81201593, 81330052)和863计划重大专项(编号：2012AA02A503, 2012AA02A209)资助

Identification of chromosomal aberration in esophageal cancer cells by mixed BAC DNA probes of chromosome arms and regions

Jiajie Hao¹, Chunli Wang^{1, 2}, Wenyue Gu^{1, 3}, Xiaoyu Cheng¹, Yu Zhang¹, Xin Xu¹, Yan Cai¹, Mingrong Wang¹

1. State Key Laboratory of Molecular Oncology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China;
2. Department of Histology and Embryology, Anhui Medical University, Hefei 230032, China;
3. Department of Pathology, Yancheng Third People’s Hospital, Yancheng 224001, China

Received:2014-01-27 Revised:2014-03-14 Online:2014-06-20 Published:2014-05-28

摘要/Abstract

摘要：

染色体畸变是恶性肿瘤细胞的重要遗传学特征, 文章旨在应用BAC DNA克隆鉴定食管癌细胞中的染色体臂和染色体区段的畸变。针对染色体各区段选取5~10个1 Mb BAC DNA, 分别混合制备成特定染色体区段的BAC DNA混合克隆, 然后将染色体臂上覆盖所有区段的上述混合克隆进一步混合制备成特定染色体臂BAC DNA混合克隆。利用简并寡核苷酸引物聚合酶链反应(Degenerate oligonucleotide primed PCR, DOP-PCR)标记染色体臂探针, 利用切口平移法(Nick translation)标记染色体区段探针, 并对食管癌细胞中期染色体进行荧光原位杂交(Fluorescence in situ hybridization, FISH)分析。正常人外周血淋巴细胞中期染色体FISH结果显示, 上述方法标记的探针具有较高的特异性。进一步利用染色体臂混合探针, 确定了多个食管癌细胞中的染色体重排所涉及的特定染色体臂; 利用染色体区段混合探针, 鉴定出KYSE140的t(1q;7q)衍生染色体中1q上的断点范围位于1q32-q41。文章成功建立了1 Mb BAC DNA混合克隆探针标记技术, 并鉴定出多个食管癌细胞中的染色体臂和染色体区段畸变, 不仅为利用M-FISH技术鉴定肿瘤细胞中的染色体畸变提供了更为准确的方法, 而且还可能进一步将该法推广应用于恶性血液病的核型分析以及产前诊断。

关键词: 染色体畸变, BAC DNA, 染色体臂和区段, 荧光原位杂交, 食管癌细胞

Abstract:

Chromosomal aberration is an important genetic feature of malignant tumor cells. This study aimed to clarify whether BAC DNA could be used to identify chromosome region and arm alterations. For each chromosome region, five to ten 1 Mb BAC DNA clones were selected to construct mixed BAC DNA clones for the particular region. All of the mixed clones from regions which could cover the whole chromosome arm were then mixed to construct mixed BAC DNA clones for the arms. Mixed BAC DNA probes of arms and regions were labeled by degenerate oligonucleotide primed PCR (DOP-PCR) and Nick translation techniques, respectively. The specificities of these probes were validated by fluorescence in situ hybridization (FISH) on the metaphase chromosomes of normal human peripheral blood lymphocytes. FISH with arm-specific mixed BAC DNA probes showed that chromosomal rearrangements and involved chromosome arms were confirmed in several esophageal cancer cells. By using region-specific mixed probes, the breakpoint on 1q from the derivative chromosome t(1q;7q) was identified in 1q32-q41 in esophageal KYSE140 cells. In conclusion, we established an effective labeling method for 1 Mb BAC DNA mixed clone probes, and chromosome arm and region rearrangements could be identified in several esophageal cancer cells by using these probes. Our study provides a more precise method for identification of chromosomal aberration by M-FISH, and the established method may also be applied to the karyotype analysis of hematological malignancies and prenatal diagnosis.

Key words: chromosomal aberration, BAC DNA, chromosome arms and regions, fluorescence hybridization, esophageal cancer cells

郝佳洁, 王春丽, 顾文跃, 程潇钰, 张钰, 徐昕, 蔡岩, 王明荣. 利用染色体区段混合BAC探针鉴定食管癌细胞中的染色体畸变[J]. 遗传, 2014, 36(6): 558-565.

Jiajie Hao, Chunli Wang, Wenyue Gu, Xiaoyu Cheng, Yu Zhang, Xin Xu, Yan Cai, Mingrong Wang. Identification of chromosomal aberration in esophageal cancer cells by mixed BAC DNA probes of chromosome arms and regions[J]. HEREDITAS(Beijing), 2014, 36(6): 558-565.

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利用染色体区段混合BAC探针鉴定食管癌细胞中的染色体畸变

Identification of chromosomal aberration in esophageal cancer cells by mixed BAC DNA probes of chromosome arms and regions

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