神经鞘氨醇磷酸胆碱对人成纤维细胞差异表达基因的筛选

Identification of differentially expressed genes in human dermal fibroblasts treated with sphingosylphosphocholine by suppression subtractive hybridizalion

目的利用抑制性消减杂交(SSH)技术构建神经鞘氨醇磷酸胆碱(SPC)对人真皮成纤维细胞的差异表达cDNA消减杂交文库,从中克隆出差异表达基因。方法原代培养人皮肤成纤维细胞；提取经SPC处理和未处理的成纤维细胞总RNA,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SPC作用于人真皮成纤维细胞的差异表达cDNA消减杂交文库,用RNA印迹验证阳性克隆,测序并登陆基因库寻找同源性基因。结果成功构建了SPC作用于人真皮成纤维细胞的差异表达cDNA文库；从实验组筛查的28个差异表达克隆中确定了6个差异表达基因,其中已知基因5个、新基因1个,分别为基质金属蛋白酶2,血小板反应蛋白1,催产素受体,锌指转录因子,铁传递蛋白受体等。结论神经鞘氨醇磷酸胆碱可上调人真皮成纤维细胞基质金属蛋白酶、血小板反应蛋白1、催产素受体、锌指转录因子、铁传递蛋白受体等基因表达。

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 suhtractive cDNA 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, thromhospondin 1, oxytocin receptor, zinc finger transcription factor-ZNF207, transferrin receptor, etc., in human dermal fibroblasts.