Abstract: Objective To construct a substractive cDNA library of human dermal fibroblasts treated with sphingosylphosphocholine (SPC) by suppression subtractive hybridization (SSH),and to clone differentially expressed genes related to SPC.Methods Primary human dermal fibroblasts were cultured in vitro.Total RNAs were extracted from the cultured fibroblasts treated with and without SPC,and reversely transcribed to cDNAs.After the cDNAs from cells treated with SPC and those from cells without SPC were hybridized with each other for two times,the hybridized products underwent two rounds of nested PCR.The PCR products were ligated with arms of T/A plasmid vectors to set up a substractive library.The positive clones were selected and verified by reverse Northern blot and DNA sequencing,and sequences obtained were analyzed for homology in Genbank.Results A subtractive eDNA library of human dermal fibroblasts treated with SPC was set up successfully with high subtractive efficiency.Six genes from twenty-eight differentially expressed clones were determined,including five known genes and one unknown gene,namely,matrix metalloprotease 2 (MMP2),thrombospondin 1 (TSP1),oxytocin receptor,zinc finger transcription factor-ZNF207,transferrin receptor (TFR),etc.Conclusions SPC could upregulate the gene expression of matrix metalloprotease 2,thrombospondin 1,oxytocin receptor,zinc finger transcription factor-ZNF207,transferrin receptor,etc.,in human dermal fibroblasts.

Key words: Fibroblasts, Gene library, Sphingosylphosphocholine, Suppression subtractive hybridization