中华皮肤科杂志 ›› 2004, Vol. 37 ›› Issue (11): 645-647.

• 论著 • 上一篇    下一篇

抑制性消减杂交筛选生长期毛乳头细胞差异表达基因

杨希川, 郝飞, 杨卫兵, 宋志强, 程波, 钟白玉, 向明明   

  1. 第三军医大学西南医院皮肤科 重庆 400038
  • 收稿日期:2003-11-11 出版日期:2004-11-15 发布日期:2004-11-15
  • 通讯作者: 郝飞, E-mail:haofei@mail.tmmu.com.cn E-mail:haofei@mail.tmmu.com.cn
  • 基金资助:
    国家自然科学基金资助项目(30200249)

Identification of Differentially Expressed Genes in Anagen Dermal Papilla by Suppression Subtractive Hybridization

YANG Xi-chuan, HAO Fei, YANG Wei-bing,SONG Zhi-qiang, CHENG Bo, ZHONG Bai-yu, XIANG Ming-ming   

  1. Department of Dermatology, Southwest Hospital of Third Military Medical University, Chongqing 400038, China
  • Received:2003-11-11 Online:2004-11-15 Published:2004-11-15

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摘要： 目的 应用抑制性消减杂交构建生长期毛囊毛乳头细胞(DPC)差异表达cDNA消减杂交文库,从中克隆鉴定出差异表达基因。方法 分别从生长期DPC及休止期DPC提取总RNA;采用SMARTcDNA合成技术合成cDNA,进行2次消减杂交及2次抑制性PCR;将产物与T/A载体连接构建生长期毛囊DPC的cDNA消减文库,通过反向RNA印迹杂交验证阳性克隆,测序并登录基因库寻找同源性基因。结果 成功构建了毛囊生长期DPC消减文库,并获得35个阳性克隆,其中功能已知基因22个,功能未知基因13个。结论 抑制性消减杂交技术是一种高效的筛选差异基因的方法,并可用于小量临床标本研究,本实验所发现的生长期DPC差异表达基因对今后研究毛囊生长调控可能具有重要意义。

关键词: 毛囊, 斑秃, 基因文库, 抑制性消减杂交

Abstract: Objectives To construct a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) and clone differentially expressed genes related to DPCs in anagen.Methods Total RNA was isolated from DPC of anagen and telogen follicles.Then ds cDNAs were synthesized in turn using SMART cDNA synthesis technique.After cDNAs from anagen and telogen follicle DPCs were hybridized with each other twice and underwent two rounds of nested PCR,PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library.Selected clones were verified by reverse Nothern blot and DNA sequencing,and the acquired sequences were analyzed for homology based on Genbank nucleotide database.Results cDNA subtractive library of DPCs in anagen follicle was set up successfully with high subtractive efficiency.Thirty-five genes were identified with 22 known functional genes and 13 unknown functional genes.Conclusions These results demonstrate the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples.Information about such alterations in gene expression might be useful for elucidating the genetic events in hair follicle growth regulation.

Key words: Hair follicle, Alopecia areata, Gene library, Suppression subtractive hybridization