Analysis Of Gene Expression In Human Dermal Fibroblasts Stimulated With Sphingosylphosphorylcholine Using CDNA Microarray And Suppesion Subtraction Hybridization

Sphingosylphosphorylcholine(SPC) is a bioactive sphingolipid metabolite. SPC has been known as a lipid pathologically accumulated in Niemann - Pick type A disease. Recently, effects of SPC on DNA synthesis and cell proliferation and mitosis-promotion were found in 3T3 fibroblasts. The role of SPC in promoting mitosis was obviously stronger than other factors, such as epidermal growth factor (EGF) and insulin. And the mitogenic action of SPC was also synergistic with EGF and insulin. SPC stimulated cell proliferation, accelerated wound healing and greatly decreased scar tissue amount in the diabetic (db/db) mice. Recently, a lot of studies have been demonstrated that SPC increased cell migration and induced the expression of plasminogen activator related to cell migration. Recent studies have shown that SPC plays important roles in tissue repairing and tissue remodeling. Wound healing is a very complicated biological process. In the this study, we mainly detected gene expression profiles in human skin fibroblasts stimulated by SPC using cDNA microarray and Suppression subtractive hybridization and investigated the mechanism of wound healing induced by SPC in gene level. At the same time, we studied signal pathway when SPC induced the expression of CTGF in human skin fibroblasts.Our results are as followed:Effects of SPC on DNA synthesis in primary cultured human skin fibroblasts(1) SPC induced an increase in DNA synthesis in primary cultured human skin fibroblasts and had dose-dependent. Increase in DNA synthesis was observed at concentration of 2 ?M, and high level of DNA synthesis at concentration of 5 ?M. But decrease in DNA synthesis was observed at concentration of 20 ?M or more due to cytotoxic effect SPC on cell. (2) SPC treatment at concentration of 5 ?M led to significantly more DNA synthesis, compared with corresponding controls at 24h, 48h, 72h after treating. The long effect of SPC on DNA synthesis may related with its half life(Its half life is 18 h).Isolation of differentially expressed genes in human skin fibroblasts stimulated by SPC using cDNA Microarray(1) A total of 194 spots survived the filtering process in cDNA microarray containing 8096 human genes. Among them, 52 genes were upregulated and 49 genes were downregulated. (2) The 101 genes differentially expressed in human skin fibroblasts treated with SPC were classified into 11 categories according to their functions. Category 1:Genes related to cells proliferation,consisting of sixgenes. Category 2:Genes related to cytoskeleton cell motility and extracellylar matrix proteins, including sixteen genes. Category 3: Genes related to transcription factors and transcription factor activity, consisting of six genes .Category 4: Gene related to Ribosome proteins, containing one gene.Category 5: Genes related to tumor suppressor, comprising of three genes. Category 6: Genes related to Immune, inflammation and cytokines, including fore genes. Category 7: Genes related to metabolism, consisting of seven genes.Category 8: Genes related to DNA binding activity and protein binding activity, including five genes.Category 9: Genes related to apoptosis and stress proteins, having one gene.Category 10: Genes related to Ion channel, consisting of five genes.Category 11:Genes related to functions, including seventeen genes.others. Among the 11 gene categories, seven of them are directly of indirectly involved in wound healing, such as CTGF,Cyr61,PA, PAI, IL-6, tubuline, actin like- 7A, transgelin, TNF-α-induced protein 6. The expression of CTGF gene was highest in all of differentially expressed genes. This result suggest that CTGF may be an important factor that downregulated the process of wound healing stimulated by SPC. Isolation of differentially expressed genes in human skin fibroblasts stimulated by SPC using SSH28 clones were found to preferentially express in a total of 264 white colonies picked up from SPC treating group. And 26 clones were detected to differentially expr...