Yang Xi-chuan, Hao Fei, Song Zhi-qiang, Cheng Bo, Yang Wei-bing, Zhong Bai-yu, Xiang Ming-ming
Department of Dermatology, Southwest Hospital of Third Military Medical University, Chongqing 400038, China.
Chin Med J (Engl). 2004 Mar;117(3):371-5.
We constructed a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) technique and clone differentially expressed genes related to DPCs in anagen.
Total mRNA was isolated from DPCs of anagen and telogen follicles. Moreover, single-strand (ss) and double-strand (ds) cDNAs were synthesized in turn using SMART PCR cDNA synthesis technology. ds cDNAs then were digested with Rsa I and divided into two groups, and ligated to the specific adaptor 1 and adaptor 2R, respectively. After cDNAs 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 demonstrated by reverse Northern blot and sequenced. The acquired sequence data were aligned against the Genbank nucleotide database.
cDNA subtractive library of DPCs in anagen follicles was set up successfully with high subtractive efficiency. Thirty-five genes were identified in this study with 22 known functional genes and 13 unknown functional genes.
All results confirm 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 could be useful for elucidating the genetic events in hair follicle growth regulation.
我们运用抑制性消减杂交（SSH）技术构建了生长期毛囊乳头细胞（DPCs）的cDNA消减文库，并克隆出生长期与DPCs相关的差异表达基因。
从生长期和休止期毛囊的DPCs中分离总mRNA。此外，依次使用SMART PCR cDNA合成技术合成单链（ss）和双链（ds）cDNA。然后用Rsa I消化ds cDNA并分为两组，分别与特异性接头1和接头2R连接。cDNA相互杂交两次并进行两轮巢式PCR。PCR产物与T/A质粒载体臂连接以构建消减文库。通过反向Northern印迹法对选定的克隆进行鉴定并测序。将获得的序列数据与Genbank核苷酸数据库进行比对。
成功构建了生长期毛囊DPCs的cDNA消减文库，消减效率高。本研究鉴定出35个基因，其中22个为已知功能基因，13个为未知功能基因。
所有结果证实了SSH在从少量临床样本中检测差异表达基因方面的有效性和敏感性。有关基因表达此类变化的信息可能有助于阐明毛囊生长调节中的遗传事件。
