应用抑制消减杂交技术克隆人大肠癌转移相关新基因

摘要/Abstract

摘要： 目的克隆新的大肠癌转移相关基因,为进一步阐述大肠癌转移的分子机制提供线索。方法利用抑制消减杂交技术,以1对来自同一亲本、转移能力不同的大肠癌细胞株SW620和SW480为研究对象,构建2种大肠癌转移促进基因和转移抑制基因的cDNA消减文库。随机挑取文库克隆进行差异筛选,对所得的差异片段进行测序和同源性分析,利用Northern Blot对新基因的表达情况进行验证。结果两种消减文库分别含有235和232个白色克隆,90%以上的白色克隆含有插入片段。随机挑取约200个阳性克隆进行差异筛选。共获得29个差异片段。测序及同源分析发现15个为未知基因,GenBank登陆号为CD485499-CD485513。其中与5号染色体序列同源的基因有6个。结论抑制消减杂交技术是筛选、克隆大肠癌转移相关新基因的有效手段;5号染色体上可能存在多个与大肠癌转移相关的新基因位点;筛选到的新基因片段为进一步克隆其全长、研究基因功能提供了实验基础。

Abstract: Objective To clone novel metastasis-associated genes of colorectal carcinoma and provide clues for the molecular mechanisms of metastasis of colorectal carcinoma. Methods By suppression subtractive hybridization (SSH), a pair of colorectal carcinoma cell lines with different metastatic potentials derived from the same parental cell line was used to construct two subtractive cDNA library specific for metastasis-accelerating genes or metastasis-suppressor genes. Partial positive clones in the two libraries were selected randomly and differentially screened, and the differentially expressed gene fragments obtained were sequenced and analyzed with BLAST software. The mRNA expressions of several new genes were confirmed by Northern blot analysis. Results Two subtractive cDNA libraries had 235 and 232 white clones respectively and more than 90% of the white clones had the inserts. About 200 positive clones selected randomly were differentially screened, and 29 differentially expressed genes were obtained. Fifteen unknown genes were found by sequence and BLAST analysis and had been collected by the GenBank dbEST database with the entry number of CIM85499 to CD485513, among which 6 novel genes were located in the fifth chromosome. Conclusion SSH is a reliable strategy for screening novel genes differentially expressed in metastasis of colorectal carcinoma. The fifth chromosome may harbor many new genes related with metastasis of colorectal carcinoma, and identified new genes can be cloned for their full length for further study of their functions.

中图分类号:

R735.3

梁莉, 丁彦青, 李欣, 杨玉芳, 肖军, 张进华, 赵培冉. 应用抑制消减杂交技术克隆人大肠癌转移相关新基因[J]. 南方医科大学学报, 2004, 24(07): 742-745.

LIANG Li, DING Yan-qing, LI Xin, YANG Yu-fang, XIAO Jun, ZHANG Jin-hua, ZHAO Pei-ran. Differential screening of novel human metastasis-associated genes of colorectal carcinoma[J]. Journal of Southern Medical University, 2004, 24(07): 742-745.

参考文献

[1] Diatchenko L, Lau YF, Campbell AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries [J]. Proc Natl Acad USA,1996, 93(12): 6025-30.
[2] 陈捷,周淑云,冯茹.抑制性消减杂交技术检测骨髓CD34+细胞基因差异表达[J].第一军医大学学报,2002,22(9):823-5.Chen J, Zhou SY, Feng R. Identification of differentially expressed genes in bone marrow CD34+ cells by suppression subtractive hybridization[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2002, 22(9): 823-5.
[3] 肖军,邹飞,蔡绍曦.抑制性消减杂交技术在研究热适应机制中的应用[J].第一军医大学学报,2003,23(10):1035-7Xiao J, Zou F, Cai SX. Application of suppression subtractive hybridization technique in the investigation of heat adaptation mechanism [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2003, 23(10): 1035-7.
[4] Hufion SE, Moerkerk PT, Brandwik R, et al. A profile of differentially expressed genes in primary colorectal cancer using suppression subtractive hybridization [J]. FEBS Lett, 1999, 463(1-2): 77-82.
[5] 王涛,陆应麟,刘爽,等.抑制消减杂交技术克隆肺癌转移调控基因[J].中华肿瘤杂志(Chin JOncol),2001,23(4):296-300.
[6] Koo TH, Lee JJ, Kim EM, et al. Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines [J]. Oncogene, 2002, 21: 4080-8.
[7] 殷剑宁,范清宇,郝新宝,等.应用抑制性消减杂交技术鉴定肿瘤转移抑制相关基因[J].中华医学遗传学杂志(Chin J Med Genet),2002,19(3):213-7.
[8] Zhang L, Cilley RE, Chinoy MR. Suppression subtractive hybridization to identify gene expressions in variant and classic small cell lung cancer cell lines[J]. J Surg Res, 2000, 93: 108-19.
[9] Sugai T, Habano W, Nakamura S, et al. Allelic losses of 17p, 5q and 18q loci in diploid and aneuploid populations of multiploid colorectal carcinomas [J]. Hum Pathol, 2000, 31 (8): 925-30.
[10] 张放,周崇治,凌云,等.散发性结直肠癌患者5号染色体杂合缺失分析[J]中华肿瘤杂志,2002,24(5):458-60
[11] Sjodahl R, Nystrom PO. Genetic aspects of colorectal cancer: the surgeon’s view [J]. Scand J Gastroenterol, 1996, 220:132-6.

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