应用抑制性消减杂交方法构建斑秃毛乳头差异表达基因文库

目的：构建斑秃区与正常毛囊毛乳头细胞（DPC）差异表达的cDNA正向和反向消减杂交文库，为从中克隆鉴定出斑秃特异性表达和生长期DPC特异性表达的基因奠定基础。方法：应用抑制性消减杂交技术，分别从斑秃区DPC及正常头皮DPC提取总mRNA；依次合成单链及双链cDNA，分别与2种不同的接头连接，再进行正向和反向的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cDNA消减文库。结论：构建成功具有高消减效率的斑秃区及正常头皮DPC cDNA消减文库，文库扩增后得到120个阳性克隆，其中90个克隆含有100－500bp插入片段，结论：应用抑制性消减杂交技术所构建的斑秃区及正常头皮DPC cDNA消减文库，为进一步批量筛选，克隆斑秃区及正常头皮DPC特异性表达的基因奠定了基础。

Construction of alopecia areata dermal papilla cDNA substractive library

Objective To construct a cDNA substractive library of dermal papilla cell (DPC) in patchy and normal area of alopecia areata with technique called suppression substractive hybridization. Methods Total mRNA was isolated form DPC of patchy and normal area. Moreover, double strand cDNAs were synthesized in turn. cDNA from patch and normal area then were restricted and divided into two groups and ligated to the specific adaptor 1 and adaptor 2 respectively. After cDNAs from patchy and normal area hybridized with each other twice and underwent two times of nested PCR, then PCR products were ligated with arms of T/A plasmid vectors to set up the substractive library. Results cDNA substractive library of DPC in patchy and normal area of alopecia areata was set up successfully with highly subtractive efficiency. The amplified library contained 120 positive clones, in which 90 positive subclones contained 100 500bp inserts. Conclusion The cDNA substractive library of DPC in patchy and normal area of alopecia areata is a highly efficient one and lays a solid foundation for screening and cloning new and specific genes from patchy and normal DPC of anagen hair follicle in DPC of alopecia areata.