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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 subtractive hybridization cDNA library of human dermal fibroblasts using sphingosine phosphate-choline (SPC) by suppression subtractive hybridization (SSH) technique, and to clone differentially expressed genes from them. Methods Human dermal fibroblasts were primary cultured. The total RNA of SPC-treated and untreated fibroblasts was extracted and cDNA was synthesized. After 2 times of subtractive hybridization and 2 times of inhibitory PCR, the product was ligated with T / A vector to construct SPC Differentially expressed cDNA clones acting on human dermal fibroblasts were subcloned into hybridization libraries, positive clones were verified by Northern blotting, and sequenced and landed in the gene pool for homology genes. Results Differentially expressed cDNA library of SPC on human dermal fibroblasts was constructed successfully. Six differentially expressed genes were identified from 28 differentially expressed clones screened by the experimental group, including 5 known genes, 1 new gene, Respectively, matrix metalloproteinase 2, thrombospondin 1, oxytocin receptors, zinc finger transcription factor, transferrin receptor. Conclusion Sphingosine phosphorylcholine up-regulates the gene expression of matrix metalloproteinases, thrombospondin 1, oxytocin receptors, zinc finger transcription factors and transferrin receptors in human dermal fibroblasts.