南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (03): 270-274.

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人膀胱癌异质性表型相关基因的筛选与鉴定

杨玉琮； 李旭； 陈葳；   

  1. 西安交通大学医学院第一附属医院实验医学中心； 西安交通大学医学院第一附属医院实验医学中心 陕西西安710061； 陕西西安710061；
  • 出版日期:2006-03-20 发布日期:2006-03-20
  • 基金资助:
    国家自然科学基金(30370660)~~

Screening and identification of heterogeneous phenotype-associated genes in human bladder cancer

YANG Yu-Cong,LI Xu,CHEN Wei Center of Laboratory Medicine,First Affiliated Hospital,Medical School of Xi’an Jiaotong University,Xi’an 710061,China   

  1. 西安交通大学医学院第一附属医院实验医学中心； 西安交通大学医学院第一附属医院实验医学中心 陕西西安710061； 陕西西安710061；
  • Online:2006-03-20 Published:2006-03-20

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摘要： 目的筛选和鉴定膀胱肿瘤异质性生物学表型相关的基因。方法以已建立的两株同一来源、不同生物学表型的膀胱移行细胞癌细胞系为材料,采用抑制性削减杂交技术筛选差异表达的基因片段,构建cDNA文库;挑取克隆进行筛选,获得差异表达片段,测序并与Genbank比对,Northern blotting验证差异片段在两株细胞系的不同表达。结果建立了两个消减文库,分别含有168和206个白色克隆,白色克隆含有约200￣700 bp的插入片段,对这些克隆进行筛选,获得35个差异表达基因片段,其中15个对应于细胞骨架蛋白、细胞代谢酶基因等已知基因,另外19个为未知功能基因,1个为新EST片段,注册Genbank(注册号为DR008207)。结论抑制性消减杂交技术是筛选、克隆差异表达基因的有效手段;keratin 7、Vimentin等细胞骨架蛋白的不同表达,与细胞形态的表型差异有关;DDH等代谢基因的不同,与细胞的药物敏感性差异相关;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。 更多还原

Abstract: Objective To screen and identify heterogeneous phenotype-associated genes of human bladder transitional cell carcinoma.Methods Subtractive cDNA libraries was established by means of suppression subtractive hybridization(SSH) on the basis of two human bladder transitional cell carcinoma cell lines(BLZ-211 and BLS-211) derived from the same patient,which had similar changes in chromosomes but different cell phenotypes(in terms of cell shape,susceptibility to 5-Fu and tumorigenic capacity).The positive clones in the library were selected for screening differentially expressed gene fragments by sequence analysis and blasting,and Northern blotting was performed to confirm the differentially expressed genes.Results The subtractive forward and reverse cDNA libraries consisted of 168 and 206 white clones containing 200-700 bp inserts.After differential screening,55 cDNA clones containing 35 different transcripts were obtained,among which 15 were identified by homology analysis as known genes(such as those coding for vimentin,keratin 7,dihydrodiol dehydrogenase and thioredoxin reductase),11 as unknown genes,and 9 as new ESTs(GenBank dbEST database accession number DY505708,DY230447-8,DR008207).Conclusion SSH is a powerful method for identifying differentialy expressed genes between different cell lines or clones,and characterization of the identified genes may provide useful information for understanding the genes responsible for different cell phenotypes.

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