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.

参考文献

[1]Speicher MR, Ballard SG, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet, 1996, 12(4): 368-375.
[2]Schrock E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garini Y, Ried T. Multicolor spectral karyo-typing of human chromosomes. Science, 1996, 273(5274): 494-497.
[3]Tchinda J, Volpert S, McNeil N, Neumann T, Kennerknecht I, Ried T, Buchner T, Serve H, Berdel WE, Horst J, Hilgenfeld E. Multicolor karyotyping in acute myeloid leukemia. Leuk Lymphoma, 2003, 44(11): 1843- 1853.
[4]Kearney L. Multiplex-FISH (M-FISH): technique, develop-ments and applications. Cytogenet Genome Res, 2006, 114(3-4): 189-198.
[5]Min BJ, Ko JM, Seo ME, Choi JS, Oh SK, Jeon J, Kim E, Moon JE, Choi IH, Lee C, Kim OH, Cho TJ, Park WY. An interstitial, apparently-balanced chromosomal insertion in the etiology of Langer-Giedion syndrome in an Asian family. Eur J Med Genet, 2013, 56(10): 561-565.
[6]Mao XY, James SY, Yanez-Munoz RJ, Chaplin T, Molloy G, Oliver RT, Young BD, Lu YJ. Rapid high-resolution karyotyping with precise identification of chromosome breakpoints. Genes Chromosomes Cancer, 2007, 46(7): 675-683.
[7]Vergult S, Van Binsbergen E, Sante T, Nowak S, Vanakker O, Claes K, Poppe B, Van der Aa N, van Roosmalen MJ, Duran K, Tavakoli-Yaraki M, Swinkels M, van den Boogaard MJ, van Haelst M, Roelens F, Speleman F, Cuppen E, Mortier G, Kloosterman WP, Menten B. Mate pair sequencing for the detection of chromosomal aberra-tions in patients with intellectual disability and congenital malformations. Eur J Hum Genet, 2014, 22(5): 652-659.
[8]Gruver AM, Rogers HJ, Cook JR, Ballif BC, Schultz RA, Batanian JR, Fesler MJ, Tubbs RR. Modified array-based comparative genomic hybridization detects cryptic and variant PML-RARA rearrangements in acute promye-locytic leukemia lacking classic translocations. Diagn Mol Pathol, 2013, 22(1): 10-21.
[9]Schluth-Bolard C, Labalme A, Cordier MP, Till M, Nadeau G, Tevissen H, Lesca G, Boutry-Kryza N, Rossignol S, Rocas D, Dubruc E, Edery P, Sanlaville D. Breakpoint mapping by next generation sequencing reveals causative gene disruption in patients carrying apparently balanced chromosome rearrangements with intellectual deficiency and/or congenital malformations. J Med Genet, 2013, 50(3): 144-150.
[10]Howarth KD, Pole JC, Beavis JC, Batty EM, Newman S, Bignell GR, Edwards PA. Large duplications at reciprocal translocation breakpoints that might be the counterpart of large deletions and could arise from stalled replication bubbles. Genome Res, 2011, 21(4): 525-534.
[11]Parkin DM, Bray FI, Devesa SS. Cancer burden in the year 2000. The global picture. Eur J Cancer, 2001, 37(Suppl. 8): S4-S66.
[12]Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin, 2011, 61(2): 69-90.
[13]Wu YP, Yang YL, Han YL, Xu X, Cai Y, Yang GZ, Wang XY, Zhan QM, Wu M, Wang MR. Identification of complex chromosome abnormalities in esophageal carcinoma cells KYSE450 by multicolor fluorescence in situ hybridiza-tion. World Chinese J Digestology, 2006, 14(8): 747-751.
[14]Wu YP, Yang YL, Yang GZ, Wang XY, Luo ML, Zhang Y, Feng YB, Xu X, Han YL, Cai Y, Zhan QM, Wu M, Dong JT, Wang MR. Identification of chromosome aberrations in esophageal cancer cell line KYSE180 by multicolor fluorescence in situ hybridization. Cancer Genet Cytogenet, 2006, 170(2): 102-107.
[15]Yang Y, Chu J, Wu Y, Luo M, Xu X, Han Y, Cai Y, Zhan Q, Wang M. Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

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

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	师明磊,赖维莉,易天红,柯潇,赵志虎. 应用荧光原位杂交技术检测康柏西普基因在CHO细胞染色体上的整合状态[J]. 遗传, 2017, 39(4): 326-332.
[2]	佘朝文，蒋向辉，宋运淳，刘伟. 玉米近缘种中玉米着丝粒序列的FISH检测[J]. 遗传, 2010, 32(3): 264-270.
[3]	汤佳立，戚大石，张俞，刘慧娟，孙健英，曹清河，马代夫，李宗芸. 荧光原位杂交技术分析栽培种甘薯(Ipomoea batatas cv.Xushu No.18)染色体[J]. 遗传, 2010, 32(2): 177-182.
[4]	谢莉，韩永华，李冬郁，曾艳华. FISH分析栽培高粱×拟高粱、甜高粱×拟高粱的染色体行为[J]. 遗传, 2009, 31(4): 420-425.
[5]	康维，姚汉清，房丽丽，蔡岩，韩亚铃，徐昕，张钰，贾雪梅，王明荣 . 食管鳞癌3、8、10、20和Y染色体的非整倍性分析[J]. 遗传, 2009, 31(3): 255-260.
[6]	郇聘，张晓军，李富花，张洋，赵翠，刘保忠，相建海. 一种栉孔扇贝丝氨酸蛋白酶基因的染色体定位及其内部SNP的研究[J]. 遗传, 2009, 31(12): 1241-1247.
[7]	陆坤，徐柱，刘朝，张学勇. St基因组中的CRW同源序列在偃麦草中的FISH分析[J]. 遗传, 2009, 31(11): 1141-1148.
[8]	卢洪涌，崔英霞，史轶超，夏欣一，杨滨，姚兵，黄宇烽. 18q部分单体患儿的细胞和分子遗传学研究[J]. 遗传, 2008, 30(8): 991-995.
[9]	裔传灯，龚志云，梁国华，王飞华，汤述翥，顾铭洪. 稻属不同染色体组着丝粒BAC克隆的分离和鉴定[J]. 遗传, 2007, 29(7): 851-858.
[10]	徐延浩，杨飞，程有林，马璐，王建波，李立家. 45S rDNA和5S rDNA在南瓜、丝瓜和冬瓜染色体上的比较定位[J]. 遗传, 2007, 29(5): 614-614―620.
[11]	廖进秋，杨瑞武，周永红，辻本壽. 波兰小麦和矮兰麦45S rDNA和5S rDNA基因位点FISH分析[J]. 遗传, 2007, 29(4): 449-454.
[12]	兰添颖，刘博，董凤平，陈瑞阳，李秀兰，陈成彬. 菠菜rDNA及端粒多色荧光原位杂交分析[J]. 遗传, 2007, 29(11): 1405-1405―1408.
[13]	韩亚玲，冯彦斌，罗曼莉，徐昕，蔡岩，王明荣. 人食管癌EC9706单克隆的分离培养及其生物学特性的研究[J]. 遗传, 2007, 29(11): 1331-1335.
[14]	李，维，葛方兰，叶德萍，雷佳红，黄敏. 家蚕细胞遗传学及其应用[J]. 遗传, 2006, 28(9): 1167-1172.
[15]	张晓云，李永全，郑克勤，周汝滨，陈小萍，廖霞. 无精和严重少精症患者的遗传缺陷分析---附2例世界首报染色体异常核型[J]. 遗传, 2006, 28(6): 646-651.